Antisense oligonucleotides for the treatment of Stargardt disease

ABSTRACT

The present invention relates to the field of medicine. In particular, it relates to novel antisense oligonucleotides that may be used in the treatment, prevention and/or delay of Stargardt disease.

This application is a 35 U.S.C. § 371 national phase application of PCT/EP2017/082627, which was filed Dec. 13, 2017, and claims the benefit of European Application No. 16203864.0, which was filed Dec. 13, 2016, and European Application No. 17189492.6, which was filed Sep. 5, 2017, all of which are incorporated herein by reference as if fully set forth.

FIELD OF THE INVENTION

The present invention relates to the field of medicine. In particular, it relates to novel antisense oligonucleotides that may be used in the treatment, prevention and/or delay of Stargardt disease.

BACKGROUND OF THE INVENTION

Autosomal recessive mutations in ABCA4 cause Stargardt disease, a progressive disorder characterized by central vision loss and often leading to complete blindness. A typical hallmark of Stargardt disease is the presence of many yellow spots (flecks) distributed throughout the fundus of the patients. The ABCA4 gene is comprised of 50 exons and encodes a protein consisting of 2273 amino acids. This protein is expressed in the outer segments of cone and rod photoreceptor cells and plays an important role in the removal of waste products following phototransduction. Besides STGD1, variants in ABCA4 can also lead to other subtypes of retinal disease ranging from bull's eye maculopathy to autosomal recessive cone-rod dystrophy (arCRD; Cremers et al, 1998; Maugeri et al, 2000) and pan-retinal dystrophies (Cremers et al, 1998; Martinez-Mir et al, 1998; Shroyer et al, 2001; Duncker et al, 2014), depending on the severity of the alleles.

Biallelic ABCA4 variants can be identified in approximately 80% of the cases with STGD1 (Allikmets et al, 1997; Fujinami et al, 2013; Lewis et al, 1999; Maugeri et al, 1999; Rivera et al, 2000; Schulz et al, 2017; Webster et al, 2001; Zernant et al, 2011; Zernant et al, 2017), and 30% of cases with arCRD (Maugeri et al, 2000), after sequencing coding regions and flanking splice sites. In general, individuals with arCRD or pan-retinal dystrophy carry two severe ABCA4 alleles, whereas individuals with STGD1 carry two moderately severe variants or a combination of a mild and a severe variant (Maugeri et al, 1999; van Driel et al, 1998). It has been hypothesized that the majority of the missing ABCA4 variants in STGD1 patients reside in intronic regions of the gene, and indeed, over the last few years, several groups have demonstrated the existence of such deep-intronic variants (Bauwens et al, 2015; Bax et al, 2015; Braun et al, 2013; Lee et al, 2016; Schulz et al, 2017; Zernant et al, 2014). In 2013, Braun and colleagues (Braun et al, 2013) described two variants in intron 30 (c.4539+2001G>A and c.4539+2028C>T, hereafter denoted M1 and M2, respectively) that supposedly could affect ABCA4 pre-mRNA splicing, yet without providing experimental evidence. M2 thus far has been identified in 13 cases (Bauwens et al, 2015; Bax et al, 2015; Braun et al, 2013; Lee et al, 2016; Schulz et al, 2017; Zernant et al, 2014). M1 has been found in 31 cases and interestingly was particularly frequent in the Dutch and Belgian populations (Bauwens et al, 2015; Bax et al, 2015; Braun et al, 2013; Lee et al, 2016; Zernant et al, 2014). In addition, we have identified several additional deep-intronic ABCA4 mutations that all lead to the insertion of pseudoexons, either by activating cryptic acceptor or splice donor sites, or by strengthening ESE motifs that are located inside the pseudoexons. These additional mutations include c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T, c.5197-557G>T.

Currently, several clinical trials for STGD1 are being conducted, employing different therapeutic strategies (http://www.clinicaltrials.gov): i) gene replacement therapy by delivering the complete ABCA4 cDNA (˜6.8 kb) via a lentiviral vector (NCT01367444 and NCT01736592); ii) subretinal transplantation of human embryonic stem cell-derived retinal pigmented epithelium cells (hESC-RPE) (NCT02445612 and NCT02941991) and iii) administration of C20-D3-retinylacetate (NCT02402660). Each of these approaches have their limitations, and so far, no efficacy data have been reported from these clinical trials.

As a considerable amount of the mutations in ABCA4 affects pre-mRNA splicing of ABCA4, they represent an attractive target for antisense oligonucleotide (AON)-based splice modulation therapy. Accordingly, there is an urge to develop AONs for splice modulation of the ABCA4 gene to enable expression of a functional ABCA4 protein in subjects suffering from Stargardt disease.

SUMMARY OF THE INVENTION

The invention provides for an antisense oligonucleotide for redirecting splicing that is:

-   -   complementary or substantially complementary to a polynucleotide         with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30,         81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof;     -   preferably complementary or substantially complementary to a         polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part         thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part         thereof     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 11 or SEQ ID NO: 31, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 12 or SEQ ID NO: 32, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 13, 16, 19, 163, 166, 169, 33,         36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209,         212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248,         251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143,         146, 149, 263, 266 and SEQ ID NO: 269, or a part thereof; and     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 14, 17, 20, 164, 167, 170, 34,         37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210,         213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249,         252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144,         147, 150, 264, and SEQ ID NO: 270, ora part thereof.

The invention further provides for an antisense oligonucleotide for redirecting splicing according to any of the preceding claims, wherein said antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 128, 131, 145, 148, 151, 265, 268 and SEQ ID NO: 271.

The invention further provides for a viral vector expressing an antisense oligonucleotide for redirecting splicing according to the invention when placed under conditions conducive to expression of the exon skipping antisense oligonucleotide.

The invention further provides for a pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to the invention or a viral vector according to the invention and a pharmaceutically acceptable excipient.

The invention further provides for the antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention and the composition according to the invention for use as a medicament.

The invention further provides for the antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention and the composition according to the invention for use in the treatment an ABCA4-related disease or condition requiring modulating splicing of ABCA4.

The invention further provides for the use of the antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention and the composition according to the invention for the preparation of a medicament.

The invention further provides for the use of the antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention and the composition according to the invention for treating an ABCA4-related disease or condition requiring modulating splicing of ABCA4.

The invention further provides for a method for modulating splicing of ABCA4 in a cell, said method comprising contacting said cell with an antisense oligonucleotide for redirecting splicing according to the invention, the vector according to the invention and the composition according to the invention.

The invention further provides for a method for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4 of an individual in need thereof, said method comprising contacting a cell of said individual with an antisense oligonucleotide for redirecting splicing according to the invention, the vector according the invention and the composition according to the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the results of example A, wherein rescue of splice defects caused by ABCA4 mutation c.4539+1100A>G and c.4539+1106C>T was accomplished by delivery of AONs in a minigene assay.

FIG. 2 depicts the results of example C wherein rescue of splice defects caused by ABCA4 mutation c.4539+2001G>A by AONs was accomplished by delivery of AONs to cultured patient-derived photoreceptor precursor cells.

FIG. 3 depicts the results of example B wherein rescue of splice defects caused by ABCA4 mutation c.4539+2001G>A by AONs was accomplished by delivery of AONs in a minigene assay.

FIG. 4 (A) depicts the results of example C wherein the splice defects caused by ABCA4 mutations c.4539+2001G>A (M1) and c.4539+2028C>T (M2) are identified. Aberrantly spliced bands were detected, especially after cycloheximide (CHX) treatment (+). Actin (ACTB) RT-PCR was used as a control.

(B) Quantification of the ratio of correctly and aberrantly spliced ABCA4 transcript for each cell line with and without CHX.

FIG. 5 In silico characterization of the effect caused by deep-intronic variants M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T). The boundaries of the 345-bp pseudoexon with the location of M1 and M2, the genomic positions of the splice sites, the splicing events detected, and the splice site predictions for both acceptor and donor sites are schematically represented. The dotted line represents the splicing from a cryptic splice donor site in exon 30 at position g.94,495,074 (GRCh37/hg19) to the normal splice acceptor site of exon 31 (r.4467_4539del, p.Cys1490Glufs*12). The predicted values of the splice acceptor and donor sites in the control and mutant situations did not show any difference. In the middle panels, the effects of the variants enhancing or creating new ESE motifs are depicted. SSFL: SpliceSiteFinder-like and HSF: Human Splicing Finder.

FIG. 6 (A) Schematic representation of the pseudoexon, indicating the location of the variants, the SC35 motifs with the highest scores and the antisense oligonucleotides (AONs). (B) RNA analysis on AON-treated cells. RT-PCR from exon 30 to exon 31 of ABCA4 in control, M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T)-containing photoreceptor precursor cells (PPCs) upon AON delivery. Actin (ACTB) mRNA amplification was used to normalize samples. NT−: non-treated and in absence of cycloheximide (CHX); NT+: non-treated in the presence of CHX; A1: AON1; A2: AON2; A3: AON3; A4: AON4; S: SON and MQ: PCR negative control. (C) Semi-quantification of the ratio of correctly versus aberrantly spliced transcripts in all M1 and M2 samples. (D) Percentage of correction of each AON compared to the NT+ based on the ratio observed in FIG. 3C. Statistical differences in the efficacy of the AONs for M1 and M2 are indicated with an asterisk (*:p≤0.05 using Mann-Whitney test).

FIG. 7 (A) Gene expression profile of one control and M1/M2-derived induced pluripotent stem cells (iPSCs) compared with the respective parental fibroblast lines.

(B) Gene expression profile of one control and M1/M2-derived photoreceptor precursor cells (PPCs) after one month of differentiation compared with iPSCs. The appearance of PPCs can be deduced by the increase in expression of CRX. The differentiation into photoreceptor-like cells is shown by the increased expression of OPN1SW, OPN1M/LW, RCV1 and ABCA4 compared with the pluripotency gene OCT3/4. The results are shown as the mean±SD. All data were plotted relative to the expression of ACTB.

FIG. 8 RT-PCR analysis from exon 2 to exon 5 of ABCA4 in control (CON), M1 (c.4539+2001G>A) and M2 (c.4539+2028C>T) photoreceptor precursor cells in the absence (−) and presence (+) of CHX. Human adult retina RNA was used as a control, while MQ was the negative control of the reaction.

FIG. 9 shows the screening of in total 26 AON sequences for their ability to correct splicing defects caused by the c.4539+2001G>A mutation. (A) Representative electrophoresis picture of an RT-PCR to detect from exon 30 to exon 31 of ABCA4 in patient-derived PPCs. The lower band represents the correct transcript while the upper bands represent the aberrant ones. The aberrant bands were detected after cycloheximide (CHX) treatment, indicating that those transcripts undergo non-sense mediated decay (NMD). Twenty-six different AON molecules were delivered to the cells together, as well as two SON (negative controls named SON1 and SON2). Results were compared to the non-treated cells (NT) in the presence of CHX (+CHX). Actin was used as loading control. MQ states for the negative control of the PCR.

Representation of the percentage of the (B) correct(ed) transcripts and the (C) aberrant transcripts after semi-quantification of two independent replicates. Based on the percentage AON molecules were classified in effective (solid grey), moderately effective (dotted pattern), poorly effective (striped pattern) and non-effective (crossed pattern). Solid, dotted and dashed lines indicate the thresholds to determine the effectiveness of the different AONs. In white are the controls indicating the basal aberrant transcript levels. In black the sample that was not treated with AON and was not subjected to CHX treatment.

FIG. 10 displays the screening of AONs for seven mutations in ABCA4 gene that cause pseudoexon inclusion. Midigenes containing the genomic region were mutagenized to insert the mutation found in humans. Subsequently these midigenes were transfected into HEK293T cells and 24 hours later different AONs were delivered to those cells. Analysis was performed by RT-PCR. For all variants three AONs were designed, and a SON was delivered as a negative control. NT states for non-treated and represents the transfected cells not subjected to AON treatment. HEK lane is an extra negative control consisting on untransfected HEK293T cells.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: Name: 1 genomic DNA ABCA4 2 cDNA ABCA4 3 Protein ABCA4 10 Pseudoexon 30-31(68) RNA 11 Pseudoexon 30-31(68) RNA; smaller target 12 Pseudoexon 30-31 (68) RNA; smaller target (AON area +10) 13 AON-1 Pseudoexon 30-31 (68) target site and flanking sequences (+10 nt) 14 AON-1 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 15 AON-1 Pseudoexon 30-31 (68) 16 AON-2 Pseudoexon 30-31 (68) target site and flanking sequences (+10 nt) 17 AON-2 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 18 AON-2 Pseudoexon 30-31 (68) 19 AON-3 Pseudoexon 30-31 (68) target site and flanking sequences (+10 nt) 20 AON-3 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 21 AON-3 Pseudoexon 30-31 (68) 30 Pseudoexon 30-31 (345) RNA 31 Pseudoexon 30-31 (345) RNA; smaller target 32 Pseudoexon 30-31 (345) RNA; smaller target (AON area +10) 33 AON-1 Pseudoexon 30-31 (345) target site and flanking sequences (+10 nt) 34 AON-1 Pseudoexon 30-31 (345) target site and flanking sequences (+5 nt) 35 AON-1 Pseudoexon 30-31 (345) 36 AON-2 Pseudoexon 30-31 (345) target site and flanking sequences (+10 nt) 37 AON-2 Pseudoexon 30-31 (345) target site and flanking sequences (+5 nt) 38 AON-2 Pseudoexon 30-31 (345) 39 AON-3 Pseudoexon 30-31 (345) target site and flanking sequences (+10 nt) 40 AON-3 Pseudoexon 30-31 (345) target site and flanking sequences (+5 nt) 41 AON-3 Pseudoexon 30-31 (345) 42 AON-4 Pseudoexon 30-31 (345) target site and flanking sequences (+10 nt) 43 AON-4 Pseudoexon 30-31 (345) target site and flanking sequences (+5 nt) 44 AON-4 Pseudoexon 30-31 (345) 45 SON-1 Pseudoexon 30-31 (345) sense version of SEQ ID NO: 35 50 pCI-Neo-Rho-ABCA4-30-31 wild type 51 pCI-Neo-Rho-ABCA4-30-31 c.4539 + 1100G 52 pCI-Neo-Rho-ABCA4-30-31 c.4539 + 1106T 53 pCI-Neo-Rho-ABCA4-30-31 c.4539 + 2001A 54 ABCA4_ex2 Fw 55 ACTB_ex3 Fw 56 ABCA4_ex30 Fw 57 ABCA4_ex20/21 Fw 58 CRX Fw 59 LIN28 Fw 60 NANOG Fw 61 OCT4 Fw 62 OPN1M/LW Fw 63 OPN1SWFw 64 RCV1 Fw 65 SOX2 Fw 66 ABCA4_ex5 Rv 67 ACTB_ex4 Rv 68 ABCA4_ex31 Rv 69 ABCA4_ex21 Rv 70 CRX Rv 71 LIN28 Rv 72 NANOG Rv 73 OCT4 Rv 74 OPN1M/LW Rv 75 OPN1SW Rv 76 RCV1 Rv 77 SOX2 Rv 80 Pseudoexon 6-7 (162) 81 Pseudoexon 6-7 (162) larger target + flanking sequences (+50 nt) 82 Pseudoexon 6-7 (162) larger target + flanking sequences (+20 nt) 83 AON-1 Pseudoexon 6-7 (162) target site and flanking sequences (+10 nt) 84 AON-1 Pseudoexon 6-7 (162) target site and flanking sequences (+5 nt) 85 AON-1 Pseudoexon 6-7 (162) 86 AON-2 Pseudoexon 6-7 (162) target site and flanking sequences (+10 nt) 87 AON-2 Pseudoexon 6-7 (162) target site and flanking sequences (+5 nt) 88 AON-2 Pseudoexon 6-7 (162) 89 AON-3 Pseudoexon 6-7 (162) target site and flanking sequences (+10 nt) 90 AON-3 Pseudoexon 6-7 (162) target site and flanking sequences (+5 nt) 91 AON-3 Pseudoexon 6-7 (162) 100 Pseudoexon 7-8 (141) 101 Pseudoexon 7-8 (141) larger target + flanking sequences (+50 nt) 102 Pseudoexon 7-8 (141) larger target + flanking sequences (+20 nt) 103 AON-1 Pseudoexon 7-8 (141) target site and flanking sequences (+10 nt) 104 AON-1 Pseudoexon 7-8 (141) target site and flanking sequences (+5 nt) 105 AON-1 Pseudoexon 7-8 (141) 106 AON-2 Pseudoexon 7-8 (141) target site and flanking sequences (+10 nt) 107 AON-2 Pseudoexon 7-8 (141) target site and flanking sequences (+5 nt) 108 AON-2 Pseudoexon 7-8 (141) 109 AON-3 Pseudoexon 7-8 (141) target site and flanking sequences (+10 nt) 110 AON-3 Pseudoexon 7-8 (141) target site and flanking sequences (+5 nt) 111 AON-3 Pseudoexon 7-8 (141) 120 Pseudoexon 7-8 (56) 121 Pseudoexon 7-8 (56) larger target + flanking sequences (+50 nt) 122 Pseudoexon 7-8 (56) larger target + flanking sequences (+20 nt) 123 AON-1 Pseudoexon 7-8 (56) target site and flanking sequences (+10 nt) 124 AON-1 Pseudoexon 7-8 (56) target site and flanking sequences (+5 nt) 125 AON-1 Pseudoexon 7-8 (56) 126 AON-2 Pseudoexon 7-8 (56) target site and flanking sequences (+10 nt) 127 AON-2 Pseudoexon 7-8 (56) target site and flanking sequences (+5 nt) 128 AON-2 Pseudoexon 7-8 (56) 129 AON-3 Pseudoexon 7-8 (56) target site and flanking sequences (+10 nt) 130 AON-3 Pseudoexon 7-8 (56) target site and flanking sequences (+5 nt) 131 AON-3 Pseudoexon 7-8 (56) 140 Pseudoexon 13-14 (134) 141 Pseudoexon 13-14 (134) larger target + flanking sequences (+50 nt) 142 Pseudoexon 13-14 (134) larger target + flanking sequences (+20 nt) 143 AON-1 Pseudoexon 13-14 (134) target site and flanking seq's (+10 nt) 144 AON-1 Pseudoexon 13-14 (134) target site and flanking seq's (+5 nt) 145 AON-1 Pseudoexon 13-14 (134) 146 AON-2 Pseudoexon 13-14 (134) target site and flanking seq's (+10 nt) 147 AON-2 Pseudoexon 13-14 (134) target site and flanking seq's (+5 nt) 148 AON-2 Pseudoexon 13-14 (134) 149 AON-3 Pseudoexon 13-14 (134) target site and flanking seq's (+10 nt) 150 AON-3 Pseudoexon 13-14 (134) target site and flanking seq's (+5 nt) 151 AON-3 Pseudoexon 13-14 (134) 160 Pseudoexon 30-31 (68) 161 Pseudoexon 30-31 (68) larger target + flanking sequences (+50 nt) 162 Pseudoexon 30-31 (68) larger target + flanking sequences (+20 nt) 163 AON-1 Pseudoexon 30-31 (68) target site and flanking seq's (+10 nt) 164 AON-1 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 165 AON-1 Pseudoexon 30-31 (68) 166 AON-2 Pseudoexon 30-31 (68) target site and flanking seq's (+10 nt) 167 AON-2 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 168 AON-2 Pseudoexon 30-31 (68) 169 AON-3 Pseudoexon 30-31 (68) target site and flanking seq's (+10 nt) 170 AON-3 Pseudoexon 30-31 (68) target site and flanking sequences (+5 nt) 171 AON-3 Pseudoexon 30-31 (68) 180 Pseudoexon 30-31 (345) 181 Pseudoexon 30-31 (345) larger target + flanking sequences (+20 nt) 182 AON-1 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 183 AON-1 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 184 AON-1 Pseudoexon 30-31 (345) 185 AON-2 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 186 AON-2 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 187 AON-2 Pseudoexon 30-31 (345) 188 AON-3 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 189 AON-3 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 190 AON-3 Pseudoexon 30-31 (345) 191 AON-4 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 192 AON-4 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 193 AON-4 Pseudoexon 30-31 (345) 194 AON-5 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 195 AON-5 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 196 AON-5 Pseudoexon 30-31 (345) 197 AON-6 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 198 AON-6 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 199 AON-6 Pseudoexon 30-31 (345) 200 AON-7 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 201 AON-7 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 202 AON-7 Pseudoexon 30-31 (345) 203 AON-8 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 204 AON-8 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 205 AON-8 Pseudoexon 30-31 (345) 206 AON-9 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 207 AON-9 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 208 AON-9 Pseudoexon 30-31 (345) 209 AON-10 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 210 AON-10 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 211 AON-10 Pseudoexon 30-31 (345) 212 AON-11 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 213 AON-11 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 214 AON-11 Pseudoexon 30-31 (345) 215 AON-12 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 216 AON-12 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 217 AON-12 Pseudoexon 30-31 (345) 218 AON-13 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 219 AON-13 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 220 AON-13 Pseudoexon 30-31 (345) 221 AON-14 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 222 AON-14 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 223 AON-14 Pseudoexon 30-31 (345) 224 AON-15 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 225 AON-15 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 226 AON-15 Pseudoexon 30-31 (345) 227 AON-16 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 228 AON-16 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 229 AON-16 Pseudoexon 30-31 (345) 230 AON-17 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 231 AON-17 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 232 AON-17 Pseudoexon 30-31 (345) 233 AON-18 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 234 AON-18 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 235 AON-18 Pseudoexon 30-31 (345) 236 AON-19 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 237 AON-19 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 238 AON-19 Pseudoexon 30-31 (345) 239 AON-20 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 240 AON-20 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 241 AON-20 Pseudoexon 30-31 (345) 242 AON-21 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 243 AON-21 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 244 AON-21 Pseudoexon 30-31 (345) 245 AON-22 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 246 AON-22 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 247 AON-22 Pseudoexon 30-31 (345) 248 AON-23 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 249 AON-23 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 250 AON-23 Pseudoexon 30-31 (345) 251 AON-24 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 252 AON-24 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 253 AON-24 Pseudoexon 30-31 (345) 254 AON-25 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 255 AON-25 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 256 AON-25 Pseudoexon 30-31 (345) 257 AON-26 Pseudoexon 30-31 (345) target site and flanking seq's (+10 nt) 258 AON-26 Pseudoexon 30-31 (345) target site and flanking seq's (+5 nt) 259 AON-26 Pseudoexon 30-31 (345) 260 Pseudoexon 36-37 (188) 261 Pseudoexon 36-37 (188) larger target + flanking sequences (+50 nt) 262 Pseudoexon 36-37 (188) larger target + flanking sequences (+20 nt) 263 AON-1 Pseudoexon 36-37 (188) target site and flanking seq's (+10 nt) 264 AON-1 Pseudoexon 36-37 (188) target site and flanking seq's (+5 nt) 265 AON-1 Pseudoexon 36-37 (188) 266 AON-2 Pseudoexon 36-37 (188) target site and flanking seq's (+10 nt) 267 AON-2 Pseudoexon 36-37 (188) target site and flanking seq's (+5 nt) 268 AON-2 Pseudoexon 36-37 (188) 269 AON-3 Pseudoexon 36-37 (188) target site and flanking seq's (+10 nt) 270 AON-3 Pseudoexon 36-37 (188) target site and flanking seq's (+5 nt) 271 AON-3 Pseudoexon 36-37 (188) 280 SON-1 (c.4539 + 2001G > A, sense version of AON1 30-31 (345)) 281 SON-2 (c.4539 + 2001G > A, sense version of AON4 30-31 (345)) 282 SON-3 (c.1937 + 435C > G, sense version of AON2 13-14 (134)) 290 pCI-Neo-Rho-ABCA4-intron6-intron7 wild type 291 pCI-Neo-Rho-ABCA4-intron6-intron7 c.769 − 784T 292 pCI-Neo-Rho-ABCA4-intron6-intron11 wild type 293 pCI-Neo-Rho-ABCA4-intron6-intron11 c.859 − 540G 294 pCI-Neo-Rho-ABCA4-intron6-intron11 c.859 − 506C 295 pCI-Neo-Rho-ABCA4-intron11-intron15 wild type 296 pCI-Neo-Rho-ABCA4-intron11-intron15 c.1937 + 435G 297 pCI-Neo-Rho-ABCA4-intron29-intron32 wild type 298 pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539 + 1100G 299 pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539 + 1106T 300 pCI-Neo-Rho-ABCA4-intron31-intron37 wild type 301 pCI-Neo-Rho-ABCA4-intron31-intron37 c.5197 − 557T 302 RHO_ex3 fw 303 ABCA4_ex7 rev 304 ABCA4_ex7 fw 305 ABCA4_ex8 rev 306 ABCA4_ex13 fw 307 ABCA4_ex14 rev 308 ABCA4_ex30 fw 309 ABCA4_ex32 rev 310 ABCA4_ex32 fw 311 ABCA4_ex37 rev

TABLE 1 Antisense oligonucleotide (AON) characteristics SEQ ID Tm Name NO: Sequence 5′→3′ Length % GC (° C.) AON-1 15/165 GUAAUCUGUUCUGGACUU 18 39 43.5 Pseudoexon 30-31 (68) AON-2 18/168 UAGAACUCCCAGGACAGG 18 56 50.3 Pseudoexon 30-31 (68) AON-3 21/171 CUAAAUCCCCCAGGAGAU 18 50 48 Pseudoexon 30-31 (68) AON-1  85 GAUGGAAUCACUGAUCCUAG 20 45 49.7 Pseudoexon 6-7 (162) AON-2  88 AGCUCCAGAGACUGAUGUGA 20 50 51.8 Pseudoexon 6-7 (162) AON-3  91 CUCACCACUGCUCCUGC 17 65 51.9 Pseudoexon 6-7 (162) AON-1 105 CCCACCAAGAUGGGGAUACU 20 55 53.8 Pseudoexon 7-8 (141) AON-2 108 GGUUCUGUUGUCCCACCAAG 20 55 53.8 Pseudoexon 7-8 (141) AON-3 111 CAAAUCACAGACUGACCCCU 20 50 51.8 Pseudoexon 7-8 (141) AON-1 125 GACUGAGCAAUACUCCGUC 19 53 51.1 Pseudoexon 7-8 (56) AON-2 128 AUCACAGAGUGACCCCUAG 19 53 51.1 Pseudoexon 7-8 (56) AON-3 131 CUGAGCAAUACUCCGUCU G 19 53 51.1 Pseudoexon 7-8 (56) AON-1 145 CUCCCAGGAACCAGACCUA 19 58 53.2 Pseudoexon 13-14 (134) AON-2 148 GCUCAUCCAACACAUUCCUC 20 50 51.8 Pseudoexon 13-14 (134) AON-3 151 CCUGGGAUGGGAGUGUC 17 65 51.9 Pseudoexon 13-14 (134) AON-1 35/184 ACAGGAG U CCUCAGCAUUG 19 53 51.1 Pseudoexon 30-31 (345) AON-2 38/187 UUUUGUCCAGGGACCAAGG 19 53 51.1 Pseudoexon 30-31 (345) AON-3 41/190 CUGUUACAUUUUGUCCAGG 19 42 46.8 Pseudoexon 30-31 (345) AON-4 44/193 GGGGCACAGAGGACUGAGA 19 63 55.4 Pseudoexon 30-31 (345) AON-5 196 GAGAGAAAAUAUUGCUUGAGAA 22 32 47.4 Pseudoexon 30-31 (345) AON-6 199 GCAGAUGAGCUGUGAUUCAA 20 45 49.7 Pseudoexon 30-31 (345) AON-7 202 UAUGAUGCAGCAGAUGAGCUG 21 48 52.4 Pseudoexon 30-31 (345) AON-8 205 UGGGAUCCCUAUGAUGCAGC 20 55 53.8 Pseudoexon 30-31 (345) AON-9 208 AGAGGACUGAGACAAGUUCC 20 50 51.8 Pseudoexon 30-31 (345) AON-10 211 GCUUCCUCUUGGGGCACAGA 20 60 55.9 Pseudoexon 30-31 (345) AON-11 214 CCUCAGCAUUGACAGCAA 18 50 48 Pseudoexon 30-31 (345) AON-12 217 ACAGGAGCCCUCAGCAUUG 19 58 53.2 Pseudoexon 30-31 (345) AON-13 220 UGGAGGCAGCCACAGGAG 18 67 54.9 Pseudoexon 30-31 (345) AON-14 223 GAUGCUGGAGGGUUUUGAGUG 21 52 54.4 Pseudoexon 30-31 (345) AON-15 226 GAUGCUGG A GAGUUUUGAGUG 21 48 52.4 Pseudoexon 30-31 (345) AON-16 229 GCCUUGACGUCCUGAUGCU 19 58 53.2 Pseudoexon 30-31 (345) AON-17 232 GCCAAGAGCUCAGGGUACAG 20 60 55.9 Pseudoexon 30-31 (345) AON-18 235 CUUGGCCUCCCCUCCCUC 18 72 57.2 Pseudoexon 30-31 (345) AON-19 238 AACACCAUGUAGGUAGGC 18 50 48  Pseudoexon 30-31 (345) AON-20 241 GUUUAGGAAAUGAAACACCAUG 22 36 49.2 Pseudoexon 30-31 (345) AON-21 244 GACCGCGUGGAAGUAAGG 18 61 52.6 Pseudoexon 30-31 (345) AON-22 247 AUAAGUUUCUAAGCUGGACAG 21 38 48.5 Pseudoexon 30-31 (345) AON-23 250 GGACCAAGGACCAACACUAC 20 55 53.8 Pseudoexon 30-31 (345) AON-24 253 GGCUGUUACAUUUUGUCCAGG 21 48 52.4 Pseudoexon 30-31 (345) AON-25 256 GGCAGGAACUGGCUUGCCUU 20 60 55.9 Pseudoexon 30-31 (345) AON-26 259 AGAAGUGAAAGAAAAUGGCAGG 22 41 51.1 Pseudoexon 30-31 (345) AON-1 265 CAGAGUUGGGCACUGUUC 18 56 50.3 Pseudoexon 36-37 (188) AON-2 268 GGCUGAUCUGGUGCAGG 17 65 51.9 Pseudoexon 36-37 (188) AON-3 271 CUUACAGGAGGCUGAUCUG 19 53 51.1 Pseudoexon 36-37 (188) SON-1 45/280 CAAUGCUGAGG A CUCCUGU 19 53 51.1 Pseudoexon Sense version of AON-1 (SEQ ID NO: 30-31 (345) 35/184) SON-2 281 UCUCAGUCCUCUGUGCCCC 19 63 55.4 Pseudoexon Sense version of AON-4 (SEQ ID NO: 30-31 (345) 44/193) SON-3 282 GAGGAAUGUGUUGGAUGAGC 20 50 51.8 Pseudoexon Sense version of AON-2 (SEQ ID NO: 13-14 (134) 148)

Some mutations are located within a pseudoexon (e.g. when the mutation creates an ESE which in turn creates the pseudoexon, the mutation will be part of the pseudoexon) The AONs designed to redirect splicing will have a mismatch in view of the wild-type sequence at the site of the mutation. This is the case for AON's with SEQ ID NO's: 35/184, 131 and 226 and for SON with SEQ ID NO: 45/280; the mutation in view of the wild-type sequence is depicted bold and underlined.

TABLE 2 Additional information on AONs for pseudoexon 30-31 (345) AON Target Type of Other # region SF2 SC35 SRp40 SRp55 region comments 5 intron 0 0 1 0 Mixed 6 acceptor 1 1 1 2 Mixed 7 acceptor 1 1 2 2 Mixed 8 PE 0 2 2 2 Mixed 9 PE 0 1 2 0 Mixed Partially overlapping with AON4 4 PE 1 3 3 0 Mixed 10 PE 2 3 1 0 Mixed Partially overlapping with AON4 11 PE 1 1 1 1 Mixed Partially overlapping with AON1 1 PE 1 1 2 2 Mixed c. 4539 + 2001G > A specific 12 PE 1 1 2 1 Mixed WT version of AON1 13 PE 0 2 2 2 Mixed Partially overlapping with AON1 14 PE 1 1 2 1 Closed/Open WT version of AON15 15 PE 1 1 2 1 Closed/Open c. 4539 + 2028C > T specific 16 PE 1 0 1 2 Mixed 17 PE 0 4 0 0 Closed/Open 18 PE 1 0 0 0 Closed/Open 19 PE 0 0 1 2 Closed/Open 20 PE 0 1 2 1 Closed/Open 21 PE 1 1 3 1 Mixed 22 PE 0 0 0 0 Mixed 23 PE 0 2 2 0 Mixed 2 PE 1 2 0 0 Mixed 3 PE 1 1 2 0 Mixed 24 PE 1 1 2 0 Mixed Equal to AON3 but 3 nt longer 25 Donor site 0 1 0 0 Mixed 26 Intron 0 2 2 0 Mixed

Table 2 describes the characteristics of 26 AONs that were tested for their efficacy to redirect PE inclusion due to the c.4539+2001G>A change. AONs are listed from 5′- to 3′-end of the pseudoexon. Column 2 lists the position relative to the PE. Columns 3 to 6 lists the number of predicted exonic splice enhancer motifs, i.e. SF2, SC35, SRp40 and SRp55 that overlap with the corresponding AON. Column 7 lists the configuration of the RNA at the position of the AONs, i.e. open, closed or a mixed configuration.

DETAILED DESCRIPTION OF THE INVENTION

By definition, AONs are substantially complementary (antisense) to their target, allowing them to bind to the corresponding pre-mRNA molecule, thereby preventing the binding of proteins essential for splicing. Usually, this lack of binding results in the skipping of the targeted exon, as the present inventors have previously shown for the c.2991+1655A>G mutation in CEP290 (Collin et al., 2012; Garanto et al., 2016). In addition, AONs may redirect the splicing machinery towards adjacent splice acceptor or donor sites. This has led the inventors to select ABCA4 mutations that may also be amenable for AON-based splice modulation therapy. These mutations are all deep-intronic variants that create novel splice acceptor, splice donor or exonic splice enhancer binding sites, and result in the inclusion of pseudoexons to the mRNA of the corresponding gene. AONs will be employed to block the recognition of (and thereby induce skipping of) the pseudoexon, thereby fully restoring the wild-type transcript and corresponding protein function. The following mutations have been selected:

-   -   c.769-784C>T. This mutation results in the insertion of a 162-nt         pseudoexon in between exons 6 and 7 of ABCA4.     -   c.859-540C>G. This mutation results in the insertion of a 141-nt         pseudoexon in between exons 7 and 8 of ABCA4.     -   c.859-506G>C. This mutation results in the insertion of a 56-nt         pseudoexon in between exons 7 and 8 of ABCA4.     -   c.1937+435C>G. This mutation results in the insertion of a         134-nt pseudoexon in between exons 13 and 14 of ABCA4.     -   c.4539+1100A>G and c.4539+1106C>T. These mutations result in the         same insertion of a 68-nt pseudoexon in between exons 30 and 31         of ABCA4 and can thus be treated with the same AONs.     -   c.4539+2001G>A and c.4539+2028C>T. These mutations result in the         same insertion of a 345-nt pseudoexon in between exons 30 and 31         of ABCA4 and can thus be treated with the same AONs.     -   c.5197-557G>T. This mutation results in the insertion of a         188-nt pseudoexon in between exons 36 and 37 of ABCA4.

The inventors have provided AONs to modulate splicing for the mutation classes depicted here above; the terms “modulate splicing” and “redirect splicing” are used herein interchangeably and encompass AON-based splice modulation therapy for the mutations depicted here above.

Accordingly, the present invention provides for an antisense oligonucleotide for redirecting splicing that is:

-   -   complementary or substantially complementary to a polynucleotide         with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30,         81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof;     -   preferably complementary or substantially complementary to a         polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part         thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part         thereof     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 11 or SEQ ID NO: 31, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 12 or SEQ ID NO: 32, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 13, 16, 19, 163, 166, 169, 33,         36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209,         212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248,         251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143,         146, 149, 263, 266 and SEQ ID NO: 269, or a part thereof; and     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 14, 17, 20, 164, 167, 170, 34,         37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210,         213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249,         252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144,         147, 150, 264, 268 and SEQ ID NO: 270, or a part thereof.

Herein, there is referred to: “SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 and SEQ ID NO: 261, or a part thereof”. In the context of the invention:

SEQ ID NO:'s 11, 12, 13, 14, 16, 17, 19, 20, 160, 162, 163, 164, 166, 167, 169 and 170 ora part thereof, are each a preferred part of SEQ ID NO: 10 and 161;

SEQ ID NO:'s 181, 180, 31, 32, 33, 34, 36, 37, 39, 40, 42, 43, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248, 251, 254, 257, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255 and 258 or a part thereof, are each a preferred part of SEQ ID NO: 30; SEQ ID NO:'s 82, 80, 83, 86, 89, 84, 87 and 90 or a part thereof, are each a preferred part of SEQ ID NO: 81; SEQ ID NO:'s 102, 100, 103, 106, 109, 104, 107 and 110 or a part thereof, are each a preferred part of SEQ ID NO: 10; SEQ ID NO:'s 122, 120, 123, 126, 129, 124, 127 and 130 or a part thereof, are each a preferred part of SEQ ID NO: 121; SEQ ID NO:'s 142, 140, 143, 146, 149, 144, 147 and 150 or a part thereof, are each a preferred part of SEQ ID NO: 141; SEQ ID NO:'s 262, 260, 263, 266, 269, 264, 267 and 270 or a part thereof, are each a preferred part of SEQ ID NO: 261.

The term exon skipping is herein defined as inducing, producing or increasing production within a cell of a mature mRNA that does not contain a particular exon that would be present in the mature mRNA without exon skipping. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mature mRNA with a molecule capable of interfering with sequences such as, for example, the (cryptic) splice donor or (cryptic) splice acceptor sequence required for allowing the enzymatic process of splicing, or with a molecule that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included in the mature mRNA; such molecules are herein referred to as exon skipping molecules. The term pre-mRNA refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template of a cell by transcription, such as in the nucleus.

The term exon retention is herein defined as inducing, producing or increasing production within a cell of a mature mRNA that does retain a particular exon that should be present in the mature mRNA without (aberrant) exon skipping. Exon retention is achieved by providing a cell expressing the pre-mRNA of said mature mRNA with an AON molecule capable of interfering with sequences such as, for example, alternative splice sites upstream or downstream of the regular splice sites. The term “antisense oligonucleotide” or “AON” is understood to refer to an oligonucleotide molecule comprising a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogenous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions.

The terms “antisense oligonucleotide”, “AON” and “oligonucleotide” are used interchangeably herein and are understood to refer to an oligonucleotide comprising an antisense sequence. Binding of an AON to its target can easily be assessed by the person skilled in the art using techniques that are known in the field such as the gel mobility shift assay as described in EP1619249. The term “substantially complementary” used in the context of the invention indicates that some mismatches in the antisense sequence are allowed as long as the functionality, i.e. inducing exon skipping or exon retention. Preferably, the complementarity is from 90% to 100%. In general this allows for 1 or 2 mismatches in an AON of 20 nucleotides or 1, 2, 3 or 4 mismatches in an AON of 40 nucleotides, or 1, 2, 3, 4, 5 or 6 mismatches in an AON of 60 nucleotides, etc. Optionally, said AON may further be tested by transfection into retina cells of patients. Skipping of an exon or retention of an exon may be assessed by RT-PCR (such as e.g. described in EP1619249). The complementary regions are preferably designed such that, when combined, they are specific for the exon in the pre-mRNA. Such specificity may be created with various lengths of complementary regions, as this depends on the actual sequences in other (pre-)mRNA molecules in the system. The risk that the AON will also be able to hybridize to one or more other pre-mRNA molecules decreases with increasing size of the AON. It is clear that AONs comprising mismatches in the region of complementarity but that retain the capacity to hybridize and/or bind to the targeted region(s) in the pre-mRNA, can be used in the invention. However, preferably at least the complementary parts do not comprise such mismatches as AONs lacking mismatches in the complementary part typically have a higher efficiency and a higher specificity than AONs having such mismatches in one or more complementary regions. It is thought, that higher hybridization strengths, (i.e. increasing number of interactions with the opposing strand) are favorable in increasing the efficiency of the process of interfering with the splicing machinery of the system.

The AON according to the invention preferably does not contain a stretch of CpG, more preferably does not contain any CpG. The presence of a CpG or a stretch of CpG in an oligonucleotide is usually associated with an increased immunogenicity of said oligonucleotide (Dorn and Kippenberger, 2008). This increased immunogenicity is undesired since it may induce damage of the tissue to be treated, i.e. the eye. Immunogenicity may be assessed in an animal model by assessing the presence of CD4+ and/or CD8+ cells and/or inflammatory mononucleocyte infiltration. Immunogenicity may also be assessed in blood of an animal or of a human being treated with an AON according to the invention by detecting the presence of a neutralizing antibody and/or an antibody recognizing said AON using a standard immunoassay known to the skilled person. An inflammatory reaction, type I-like interferon production, IL-12 production and/or an increase in immunogenicity may be assessed by detecting the presence or an increasing amount of a neutralizing antibody or an antibody recognizing said AON using a standard immunoassay. The AON according to the invention furthermore preferably has acceptable RNA binding kinetics and/or thermodynamic properties. The RNA binding kinetics and/or thermodynamic properties are at least in part determined by the melting temperature of an oligonucleotide (Tm; calculated with the oligonucleotide properties calculator (www.unc.edu/-cail/biotool/oligo/index) for single stranded RNA using the basic Tm and the nearest neighbor model), and/or the free energy of the AON-target exon complex (using RNA structure version 4.5). If a Tm is too high, the AON is expected to be less specific. An acceptable Tm and free energy depend on the sequence of the AON. Therefore, it is difficult to give preferred ranges for each of these parameters. An acceptable Tm may be ranged between 35 and 70° C. and an acceptable free energy may be ranged between 15 and 45 kcal/mol. The skilled person may therefore first choose an AON as a potential therapeutic compound as binding and/or being complementary to SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof as defined later herein. The skilled person may check that said AON is able to bind to said sequences as earlier defined herein. Optionally in a second step, he may use the invention to further optimize said AON by checking for the absence of CpG and/or by optimizing its Tm and/or free energy of the AON-target complex. He may try to design an AON wherein few, preferably, no CpG and/or wherein a more acceptable Tm and/or free energy are obtained by choosing a distinct sequence of ABCA4 (including SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 and SEQ ID NO: 261, or a part thereof) to which the AON is complementary. Alternatively, if an AON complementary to a given stretch within SEQ ID NO: 10 or 30, comprises a CpG, and/or does not have an acceptable Tm and/or free energy, the skilled person may improve any of these parameters by decreasing the length of the AON, and/or by choosing a distinct stretch within any of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261 to which the AON is complementary and/or by altering the chemistry of the AON.

An AON according to the invention is said to induce exon skipping if the skipping percentage as measured by real-time quantitative RT-PCR analysis is at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%. An AON according to the invention is said to induce exon retention if the retention percentage as measured by real-time quantitative RT-PCR analysis is at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100%. Preferably, an AON according to the invention comprising a part that is (substantially) complementary to SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, or a part thereof, is an AON wherein the (substantially) complementary part is at least 50% of the length of the AON according to the invention, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% or even more preferably at least 95%, or even more preferably 98% or even more preferably at least 99%, or even more preferably 100%. Preferably, an AON according to the invention comprises or consists of a sequence that is complementary or substantially complementary to a part of SEQ ID NO: 10 or 30. As an example, an AON may comprise a sequence that is complementary or substantially complementary to a part of SEQ ID NO: 10 or 30 and comprise additional flanking sequences. Preferably, an AON according to the invention is an AON wherein the part that is (substantially) complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID NO: 30, or a part thereof, comprises at least one ESE (exon splice enhancer) motif, preferably two, three, four or more ESE motifs. ESE motifs are known to the person skilled in the art. Identification and determination of an ESE is preferably performed as in the examples herein. In an embodiment, an AON according to the invention does not comprise an ESE motif.

Preferably, an AON according to the invention is an AON wherein the part that is (substantially) complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, has a length of from about 8 to about 40 nucleotides, such as preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, an AON according to the invention is an AON wherein the part that is (substantially) complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, has a length of from 8 to 40 nucleotides, such as preferably from 10 to 40 nucleotides, more preferably from 14 to 30 nucleotides, more preferably from 16 to 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, an AON according to the invention is an AON wherein the part that is (substantially) complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, or a part thereof, has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides and said part that is (substantially) complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30, 81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof, or a part thereof, has a length of at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

Additional sequences the (substantially) complementary part may be used to modify the binding of a protein, such as a splice-promoting factor, to the AON, or to modify a thermodynamic property of the AON, such as to modify target RNA binding affinity.

A preferred AON for redirecting splicing according to the invention has a length of from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. A more preferred AON for redirecting splicing according to the invention has a length of from 8 to 100 nucleotides, preferably from 10 to 40 nucleotides, more preferably from 14 to 30 nucleotides, more preferably from 16 to 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. Preferably, an AON according to the invention has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. Preferably, an AON according to the invention has a length of at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

In an embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 128, 131, 145, 148, 151, 265, 268 and SEQ ID NO: 271.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 15. The preferred AON comprising SEQ ID NO: 15 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 18. The preferred AON comprising SEQ ID NO: 18 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 21. The preferred AON comprising SEQ ID NO: 21 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 35. The preferred AON comprising SEQ ID NO: 35 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 38. The preferred AON comprising SEQ ID NO: 38 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 41. The preferred AON comprising SEQ ID NO: 41 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 44. The preferred AON comprising SEQ ID NO: 44 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 165. The preferred AON comprising SEQ ID NO: 165 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 168. The preferred AON comprising SEQ ID NO: 168 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 171. The preferred AON comprising SEQ ID NO: 171 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 184. The preferred AON comprising SEQ ID NO: 184 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 187. The preferred AON comprising SEQ ID NO: 187 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 190. The preferred AON comprising SEQ ID NO: 190 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 193. The preferred AON comprising SEQ ID NO: 193 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 196. The preferred AON comprising SEQ ID NO: 196 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 199. The preferred AON comprising SEQ ID NO: 199 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 202. The preferred AON comprising SEQ ID NO: 202 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 205. The preferred AON comprising SEQ ID NO: 205 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 208. The preferred AON comprising SEQ ID NO: 208 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 211 The preferred AON comprising SEQ ID NO: 211 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 214. The preferred AON comprising SEQ ID NO: 214 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 217. The preferred AON comprising SEQ ID NO: 217 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 220. The preferred AON comprising SEQ ID NO: 220 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 223. The preferred AON comprising SEQ ID NO: 223 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 226. The preferred AON comprising SEQ ID NO: 226 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 229. The preferred AON comprising SEQ ID NO: 229 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 232. The preferred AON comprising SEQ ID NO: 232 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 235. The preferred AON comprising SEQ ID NO: 235 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 238. The preferred AON comprising SEQ ID NO: 238 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 241. The preferred AON comprising SEQ ID NO: 241 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 244. The preferred AON comprising SEQ ID NO: 244 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 247. The preferred AON comprising SEQ ID NO: 247 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 250. The preferred AON comprising SEQ ID NO: 250 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 253. The preferred AON comprising SEQ ID NO: 253 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 256. The preferred AON comprising SEQ ID NO: 256 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 259. The preferred AON comprising SEQ ID NO: 259 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 85. The preferred AON comprising SEQ ID NO: 85 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 88. The preferred AON comprising SEQ ID NO: 88 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 91. The preferred AON comprising SEQ ID NO: 91 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 105. The preferred AON comprising SEQ ID NO: 105 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 108. The preferred AON comprising SEQ ID NO: 108 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 111. The preferred AON comprising SEQ ID NO: 111 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 125. The preferred AON comprising SEQ ID NO: 125 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 128. The preferred AON comprising SEQ ID NO: 128 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 131. The preferred AON comprising SEQ ID NO: 131 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 145. The preferred AON comprising SEQ ID NO: 145 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 148. The preferred AON comprising SEQ ID NO: 148 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 151. The preferred AON comprising SEQ ID NO: 151 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 265. The preferred AON comprising SEQ ID NO: 265 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 268. The preferred AON comprising SEQ ID NO: 268 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

In a preferred embodiment, there is provided an AON comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 271. The preferred AON comprising SEQ ID NO: 271 preferably comprises from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, or preferably comprises or consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

An AON for redirecting splicing according to the invention may comprise one of more RNA residue (ribonucleotide), or one or more DNA residue (deoxyribonucleotide), and/or one or more nucleotide analogues or equivalents, as will be further detailed herein below.

It is preferred that an AON for redirecting splicing according to the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the antisense oligonucleotide for the target sequence. Therefore, in a preferred embodiment, the AON comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents.

Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report, demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

It is further preferred that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.

A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen et al., 1991). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar, 2005). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al., 1993). A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent according to the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl phosphonate including 3′-alkylene phosphonate, 5′-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3′-amino phosphoramidate and am inoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.

A further preferred nucleotide analogue or equivalent according to the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2′, 3′ and/or 5′ position such as a —OH; —F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative of. A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), in which the 2′-carbon atom is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2′-O, 4′-C-ethylene-bridged nucleic acid (Morita et al., 2001). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA. In another embodiment, a nucleotide analogue or equivalent according to the invention comprises one or more base modifications or substitutions. Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.

It is understood by a skilled person that it is not necessary for all positions in an AON to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single AON or even at a single position within an AON. In certain embodiments, an AON according to the invention has at least two different types of analogues or equivalents. Accordingly, a preferred AON for redirecting splicing according to the invention comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.

It will also be understood by a skilled person that different AON's according to the invention can be combined for efficient therapy. In an embodiment, a combination of at least two AON's according to the invention are used, such as two different AON's according to the invention, three different AON's according to the invention, four different AON's according to the invention, or five AON's according to the invention.

An AON for redirecting splicing according to the invention can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells, preferably retina cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.

An AON for redirecting splicing according to the invention may be indirectly administrated using suitable means known in the art. It may for example be provided to an individual or a cell, tissue or organ of said individual as such, as a so-called ‘naked’ AON. It may also be administered in the form of an expression vector wherein the expression vector encodes an RNA transcript comprising the sequence of said AON according to the invention. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of an AON for redirecting splicing according to the invention. Accordingly, the invention provides for a viral vector expressing an antisense oligonucleotide for redirecting splicing according to the invention when placed under conditions conducive to expression of the antisense oligonucleotide for redirecting splicing. A cell can be provided with an AON for redirecting splicing according to the invention by plasmid-derived antisense oligonucleotide expression or viral expression provided by adenovirus- or adeno-associated virus-based vectors. Expression may be driven by an RNA polymerase II promoter (Pol II) such as a U7 RNA promoter or an RNA polymerase III (Pol III) promoter, such as a U6 RNA promoter. A preferred delivery vehicle is a viral vector such as an adeno-associated virus vector (AAV), or a retroviral vector such as a lentivirus vector and the like. Also, plasmids, artificial chromosomes, plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an AON for redirecting splicing according to the invention. Preferred for the invention are those vectors wherein transcription is driven from Pall promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are Pall driven transcripts, preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such fusions may be generated as previously described (Gorman et al., 1998).

A preferred expression system for an AON for redirecting splicing according to the invention is an adenovirus associated virus (AAV)-based vector. Single chain and double chain AAV-based vectors have been developed that can be used for prolonged expression of antisense nucleotide sequences for highly efficient redirection of splicing. A preferred AAV-based vector, for instance, comprises an expression cassette that is driven by an RNA polymerase III-promoter (Pol III) or an RNA polymerase II promoter (Pol II). A preferred RNA promoter is, for example, a Pol III U6 RNA promoter, or a Pol II U7 RNA promoter.

The invention accordingly provides for a viral-based vector, comprising a Pol II or a Pol III promoter driven expression cassette for expression of an AON for redirecting splicing according to the invention.

An AAV vector according to the invention is a recombinant AAV vector and refers to an AAV vector comprising part of an AAV genome comprising an encoded AON for redirecting splicing according to the invention encapsidated in a protein shell of capsid protein derived from an AAV serotype as depicted elsewhere herein. Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, AAV9 and others. A protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1, 2, 3, 4, 5, 8, 9 and others. A protein shell may also be named a capsid protein shell. AAV vector may have one or preferably all wild type AAV genes deleted, but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell. In the context of the invention a capsid protein shell may be of a different serotype than the AAV vector genome ITR. An AAV vector according to present the invention may thus be composed of a capsid protein shell, i.e. the icosahedral capsid, which comprises capsid proteins (VP1, VP2, and/or VP3) of one AAV serotype, e.g. AAV serotype 2, whereas the ITRs sequences contained in that AAV5 vector may be any of the AAV serotypes described above, including an AAV2 vector. An “AAV2 vector” thus comprises a capsid protein shell of AAV serotype 2, while e.g. an “AAV5 vector” comprises a capsid protein shell of AAV serotype 5, whereby either may encapsidate any AAV vector genome ITR according to the invention.

Preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2, 5, 8 or AAV serotype 9 wherein the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9; such AAV vector is referred to as an AAV2/2, AAV2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV 5/9, AAV8/2, AAV 8/5, AAV8/8, AAV8/9, AAV9/2, AAV9/5, AAV9/8, or an AAV9/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 5; such vector is referred to as an AAV 2/5 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 8; such vector is referred to as an AAV 2/8 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 9; such vector is referred to as an AAV 2/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 2; such vector is referred to as an AAV 2/2 vector.

A nucleic acid molecule encoding an AON for redirecting splicing according to the invention represented by a nucleic acid sequence of choice is preferably inserted between the AAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3′ termination sequence. “AAV helper functions” generally refers to the corresponding AAV functions required for AAV replication and packaging supplied to the AAV vector in trans. AAV helper functions complement the AAV functions which are missing in the AAV vector, but they lack AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region or functional substantially identical sequences thereof. Rep and Cap regions are well known in the art, see e.g. (Chiorini et al., 1999) or U.S. Pat. No. 5,139,941, incorporated herein by reference. The AAV helper functions can be supplied on an AAV helper construct, which may be a plasmid. Introduction of the helper construct into the host cell can occur e.g. by transformation, transfection, or transduction prior to or concurrently with the introduction of the AAV genome present in the AAV vector as identified herein. The AAV helper constructs according to the invention may thus be chosen such that they produce the desired combination of serotypes for the AAV vector's capsid protein shell on the one hand and for the AAV genome present in said AAV vector replication and packaging on the other hand.

“AAV helper virus” provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as described in U.S. Pat. No. 6,531,456 incorporated herein by reference.

Preferably, an AAV genome as present in a recombinant AAV vector according to the invention does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. An AAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art.

Preferably, an AAV vector according to the invention is constructed and produced according to the method according to Garanto et al., 2016 which is herein incorporated by reference.

A preferred AAV vector according to the invention is an AAV vector, preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, expressing an AON for redirecting splicing according to the invention that is an AON that comprises, or preferably consists of, a sequence that is:

-   -   complementary or substantially complementary to a polynucleotide         with a nucleotide sequence consisting of SEQ ID NO: 10, 161, 30,         81, 101, 121, 141 or SEQ ID NO: 261, or a part thereof;     -   preferably complementary or substantially complementary to a         polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 162, 181, 82, 102, 122, 142 or SEQ ID NO: 262, or a part         thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 160, 180, 80, 100, 120, 140 or SEQ ID NO: 260, or a part         thereof     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 11 or SEQ ID NO: 31, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 12 or SEQ ID NO: 32, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 13, 16, 19, 163, 166, 169, 33,         36, 39, 42, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209,         212, 215, 218, 221, 224, 227, 230, 233, 236, 239, 242, 245, 248,         251, 254, 257, 83, 86, 89, 103, 106, 109, 123, 126, 129, 143,         146, 149, 263, 266 and SEQ ID NO: 269, or a part thereof; and     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 14, 17, 20, 164, 167, 170, 34,         37, 40, 43, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210,         213, 216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249,         252, 255, 258, 84, 87, 90, 104, 107, 110, 124, 127, 130, 144,         147, 150, 264, 268 and SEQ ID NO: 270, or a part thereof.

A further preferred AAV vector according to the invention is an AAV vector, preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, expressing an exon skipping molecule or an exon 12 retention molecule according to the invention that is expressing an AON for redirecting splicing according to the invention that comprises, or preferably consists of, a sequence selected from the group consisting of SEQ ID NO: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 85, 88, 91, 105, 108, 111, 125, 128, 131, 145, 148, 151, 265, 268 and SEQ ID NO: 271. Improvements in means for providing an individual or a cell, tissue, organ of said individual with an AON for redirecting splicing according to the invention, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method according to the invention. An AON for redirecting splicing according to the invention can be delivered as such as a ‘naked’ AON to an individual, a cell, tissue or organ of said individual. When administering an AON for redirecting splicing according to the invention, it is preferred that the molecule is dissolved in a solution that is compatible with the delivery method. Retina cells can be provided with a plasmid for antisense oligonucleotide expression by providing the plasmid in an aqueous solution. Alternatively, a preferred delivery method for an AON for redirecting splicing or a plasmid for expression of such AON is a viral vector or are nanoparticles. Preferably, viral vectors or nanoparticles are delivered to retina or other relevant cells. Such delivery to retina cells or other relevant cells may be in vivo, in vitro or ex vivo; see e.g. Garanto et al, 2016, which is herein incorporated by reference.

Alternatively, a plasmid can be provided by transfection using known transfection agents. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient or transfection agents that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell, preferably a retina cell. Preferred are excipients or transfection agents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agentia comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), Lipofectin™, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constitutent as defined herein to a cell, preferably a retina cell. Such excipients have been shown to efficiently deliver an oligonucleotide such as AON's to a wide variety of cultured cells, including retina cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity.

Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidylethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles.

Polycations such as diethylaminoethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver each constituent as defined herein, preferably an AON according to the invention, across cell membranes into cells.

In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an oligonucleotide. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an exon skipping molecule for use in the current invention to deliver it for the prevention, treatment or delay of ABCA4-related disease or condition. “Prevention, treatment or delay of an ABCA4-related disease or condition” is herein preferably defined as preventing, halting, ceasing the progression of, or reversing partial or complete visual impairment or blindness that is caused by a genetic defect in the ABCA4 gene.

In addition, an AON for redirecting splicing according to the invention could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognizing cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes.

Therefore, in a preferred embodiment, an AON for redirecting splicing according to the invention is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery.

It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be suitably formulated in one single combination or composition or preparation. Depending on their identity and specific features, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an AON for redirecting splicing according to the invention and a further adjunct compound as later defined herein.

If required and/or if desired, an AON for redirecting splicing according to the invention or a vector, preferably a viral vector, according to the invention, expressing an AON for redirecting splicing according to the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier.

Accordingly, the invention also provides for a composition, preferably a pharmaceutical composition, comprising an AON for redirecting splicing according to the invention, or a viral vector according to the invention and a pharmaceutically acceptable excipient. Such composition may comprise a single AON for redirecting splicing or viral vector according to the invention, but may also comprise multiple, distinct AON's for redirecting splicing or viral vectors according to the invention. Such a pharmaceutical composition may comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer and/or diluent may for instance be found in Remington, 2000. Each feature of said composition has earlier been defined herein.

A preferred route of administration is through intra-vitreal injection of an aqueous solution or specially adapted formulation for intraocular administration. EP2425 814 discloses an oil in water emulsion especially adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. This emulsion is less dense than the vitreous fluid, so that the emulsion floats on top of the vitreous, avoiding that the injected drug impairs vision.

If multiple distinct AON's for redirecting splicing according to the invention are used, the concentration or dose defined herein may refer to the total concentration or dose of all oligonucleotides used or the concentration or dose of each exon skipping molecule used or added. Therefore, in an embodiment, there is provided a composition wherein each or the total amount of AON's for redirecting splicing according to the invention used is dosed in an amount ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg per eye. A suitable intravitreal dose is provided and comprises between 0.05 mg and 5 mg, preferably between 0.1 and 1 mg per eye, such as about per eye: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg.

A preferred AON for redirecting splicing according to the invention, is for the treatment of an ABCA4-related disease or condition of an individual. In all embodiments of the invention, the term “treatment” is understood to include the prevention and/or delay of the ABCA4-related disease or condition. An individual, which may be treated using an AON for redirecting splicing according to the invention may already have been diagnosed as having an ABCA4-related disease or condition. Alternatively, an individual which may be treated using an AON for redirecting splicing according to the invention may not have yet been diagnosed as having a ABCA4-related disease or condition but may be an individual having an increased risk of developing a ABCA4-related disease or condition in the future given his or her genetic background. A preferred individual is a human being. In all embodiments of the invention, the ABCA4-related disease or condition is preferably Stargardt disease.

Accordingly, the invention further provides for an AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a (pharmaceutical) composition according to the invention for use as a medicament, preferably as a medicament for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4 and for use as a medicament for the prevention, treatment or delay of an ABCA4-related disease or condition. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for, a method of treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4, comprising said method comprising contacting a cell of said individual with an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for the preparation of a medicament. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for the preparation of a medicament for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

The invention further provides for the use of an AON for redirecting splicing according to the invention, a vector according to the invention or a (pharmaceutical) composition according to the invention for treating an ABCA4 related disease or condition requiring modulating splicing of ABCA4. Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.

Treatment in a use or in a method according to the invention is preferably at least once, and preferably lasts at least one week, one month, several months, one year, 2, 3, 4, 5, 6 years or longer, such as life-long. Each AON for redirecting splicing according to the invention or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing an ABCA4-related disease or condition, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an AON, composition, compound or adjunct compound according to the invention may depend on several parameters such as the severity of the disease, the age of the patient, the mutation of the patient, the number of AON for redirecting splicing according to the invention (i.e. dose), the formulation of the AON, composition, compound or adjunct compound according to the invention, the route of administration and so forth. The frequency of administration may vary between daily, weekly, at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.

Dose ranges of an AON, composition, compound or adjunct compound according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. An AON according to the invention may be used at a dose which is ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg. A suitable intravitreal dose would be between 0.05 mg and 5 mg, preferably between 0.1 and 1 mg per eye, such as about per eye: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg.

In a preferred embodiment, a concentration of an oligonucleotide as defined herein, which is ranged from 0.1 nM and 1 μM is used. Preferably, this range is for in vitro use in a cellular model such as retina cells or retinal tissue. More preferably, the concentration used is ranged from 1 to 400 nM, even more preferably from 10 to 200 nM, even more preferably from 50 to 100 nM. If multiple distinct AONs are used, this concentration or dose may refer to the total concentration or dose of the AONs or the concentration or the dose of each AON added.

In a preferred embodiment, a viral vector, preferably an AAV vector as described earlier herein, as delivery vehicle for a molecule according to the invention, is administered in a dose ranging from 1×10⁹-1×10¹⁷ virus particles per injection, more preferably from 1×10¹⁰-1×10¹² virus particles per injection.

The ranges of concentration or dose of AONs as depicted above are preferred concentrations or doses for in vivo, in vitro or ex vivo uses. The skilled person will understand that depending on the AONs used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of AONs used may further vary and may need to be optimized any further.

An AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention for use according to the invention may be administered to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing a ABCA4-related disease or condition, and may be administered in vivo, ex vivo or in vitro. An AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual already affected by or at risk of developing a ABCA4-related disease or condition, and may be administered directly or indirectly in vivo, ex vivo or in vitro. As Stargardt disease has a pronounced phenotype in retina cells, it is preferred that said targeted cells are retina cells, it is further preferred that said tissue is the retina and it is further preferred that said organ comprises or consists of the eye.

The invention further provides for a method for modulating splicing of ABCA4 in a cell comprising contacting the cell, preferably a retina cell, with an AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a (pharmaceutical) composition according to the invention. The features of this aspect are preferably those defined earlier herein. Contacting the cell with an AON for redirecting splicing according to the invention, or a viral vector according to the invention, or a composition according to the invention may be performed by any method known by the person skilled in the art. Use of the methods for delivery of AONs for redirecting splicing, viral vectors and compositions as described earlier herein is included. Contacting may be directly or indirectly and may be in vivo, ex vivo or in vitro.

Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein.

Definitions

In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 5% of the value.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the polypeptide obtainable by expression of the gene present in SEQ ID NO: 1 containing the nucleic acid sequence coding for the polypeptide should prevail.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

Embodiments of the Invention

1. An antisense oligonucleotide for redirecting splicing that is:

-   -   complementary or substantially complementary to a polynucleotide         with a nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID         NO: 30, or a part thereof;     -   preferably complementary or substantially complementary to a         polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 11 or SEQ ID NO: 31, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence consisting of SEQ ID         NO: 12 or SEQ ID NO: 32, or a part thereof;     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19,         SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39 and SEQ ID NO: 42,         or a part thereof; and     -   more preferably complementary or substantially complementary to         a polynucleotide with a nucleotide sequence selected from the         group consisting of SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20,         SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40 and SEQ ID NO: 43,         or a part thereof.

2. An antisense oligonucleotide for redirecting splicing according to embodiment 1, wherein the part that is complementary or substantially complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10 or SEQ ID NO: 30, or a part thereof, has a length of from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

3. An antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments that has a length of from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

4. An antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments, wherein said antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41 and SEQ ID NO: 44.

5. An antisense oligonucleotide for redirecting splicing according to any one of the preceding embodiments, comprising at least one ribonucleotide.

6. An antisense oligonucleotide for redirecting splicing according to any one of the preceding embodiments, comprising at least one ESE (exon splice enhancer) motif.

7. An antisense oligonucleotide for redirecting splicing according to any one of the preceding embodiments comprising a 2′-0 alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.

8. A viral vector expressing an antisense oligonucleotide for redirecting splicing according to any of the preceding embodiments when placed under conditions conducive to expression of the exon skipping antisense oligonucleotide.

9. A pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-7 or a viral vector according to embodiment 7 and a pharmaceutically acceptable excipient.

10. A pharmaceutical composition according to embodiment 9, wherein the pharmaceutical composition is for intravitreal administration and is dosed in an amount ranged from 0.05 mg and 5 mg of total antisense oligonucleotides for redirecting splicing per eye.

11. A pharmaceutical composition according to embodiment 10, wherein the pharmaceutical composition is for intravitreal administration and is dosed in an amount ranged from 0.1 and 1 mg of total antisense oligonucleotides for redirecting splicing per eye, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg of total antisense oligonucleotides for redirecting splicing per eye.

12. The antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-7, the vector according to embodiment 8 or the composition according to any one of embodiments 9-11 for use as a medicament.

13. The antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-7, the vector according to embodiment 9 or the composition according to any one of embodiments 9-11 for use in the treatment an ABCA4-related disease or condition requiring modulating splicing of ABCA4.

14. Use of the antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-7, the vector according to embodiment 8 or the composition according to any one of embodiments 9-11 for the preparation of a medicament.

15. Use of the antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-6, the vector according to embodiment 7 or the composition according to any one of embodiments 8-10 for the preparation of medicament for treating an ABCA4-related disease or condition requiring modulating splicing of ABCA4.

16. Use of the antisense oligonucleotide for redirecting splicing according to any one of embodiments 1-7, the vector according to embodiment 7 or the composition according to any one of embodiments 9-11 for treating an ABCA4-related disease or condition requiring modulating splicing of ABCA4.

17. A method for modulating splicing of ABCA4 in a cell, said method comprising contacting said cell with an antisense oligonucleotide for redirecting splicing as defined in any one of embodiments 1-7, the vector according to embodiment 7 or the composition according to any one of embodiments 9-11.

18. A method for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4 of an individual in need thereof, said method comprising contacting a cell of said individual with an antisense oligonucleotide for redirecting splicing as defined in any one of embodiments 1-7, the vector according to embodiment 7 or the composition according to any one of embodiments 9-11.

19. The antisense oligonucleotide for redirecting splicing for use according to embodiment 12 or 13, the use according to embodiment 15 or 16 or the method according to embodiment 18, wherein the ABCA4-related disease or condition is Stargardt disease.

Examples

Initially, we have assessed the in vitro efficacy of a number of AONs to redirect splice defects due to the c.4539+1100A>G, c.4539+1106C>T and c.4539+2001G>A mutations in ABCA4, in human embryonic kidney (HEK293T) cells. For this, we used minigene constructs, i.e. plasmids that harbour the sequence of a part of the ABCA4 gene, usually the region of interest with or without the mutation, and flanked by at least 500 bp of wild-type ABCA4 sequence on each side. The plasmid also contains the exonic sequences and intron-exon boundaries of exons 3 and 5 of the RHO gene on each side of the ABCA4 sequence, respectively. In this way, the effect of the ABCA4 variant on the splicing of the corresponding exon or pseudoexons can be readily measured. Later on, we used larger constructs (coined midigenes) to assess the nature of other deep-intronic variants that were discovered, including c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T. The generation of these midigenes is described in Sangermano et al. (2018) Finally, in addition to the minigene assays, we also used photoreceptor precursor cells (PPCs) from a patient with compound heterozygous ABCA4 mutations, namely the c.4539+2001G>A mutation together with the c.4892T>C (p.Leu1631Pro) on the other allele, to assess the potential of AONs to rescue the splice defect. PPCs were also used to assess the potential of AONs to rescue splice defects from a patient carrying a complex allele containing c.302+68C>T and c.4539+2028C>T (M2), and the deletion c.6148-698_6670 delinsTGTGCACCTCCCTAG on the other allele (Lee et al. 2016). First, in the Materials and Methods section, the experimental details are described, whereas the results are described and illustrated in the Results section further below.

Materials and Methods

A) Mutations: c.4539+1100A>G & c.4539+1106C>T—Minigenes

Generation of a Minigene for Each Mutation

A minigene was created including part of the intron 29, the complete exon 30, intron 30 and exon 31, and part of intron 31. This genomic region was cloned into a pCI-Neo-Rhodopsin vector using the Gateway System. The resulting vector (coined pCI-Neo-Rho-ABCA4-30-31 wild-type, SEQ ID NO: 50) was used to introduce the c.4539+1100A>G and c.4539+1106C>T mutations by site-directed mutagenesis (new vector was coined pCI-Neo-Rho-ABCA4-c.4539+1100G, SEQ ID NO: 51 and pCI-Neo-Rho-ABCA4-c.4539+1106T, SEQ ID NO: 52). The control and mutated vectors were validated by Sanger sequencing. The minigenes were then transfected in HEK293T cells, which were harvested 48 h post-transfection and were subjected to RT-PCR analysis in order to detect the splicing defect.

AON Design and Testing

The RNA analysis of the HEK293T cells transfected with the minigenes, showed the pre-mRNA splicing defect that consisted of the insertion of a pseudoexon. Using the sequence of this pseudoexon several AONs were designed. Subsequently, AONs were transfected into HEK293T together with the minigenes. To validate the AON efficacy, cells were subjected to RT-PCR analysis. The efficiency of each of the AONs was assessed by delivering identical amounts of minigene and various concentrations of AON and performing RT-PCR analysis afterwards.

RT-PCR Analysis

Total RNA was isolated by using the NucleoSpin RNA Clean-up Kit (catalog no., 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturers protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the iScript cDNA synthesis kit (catalog no., 1708891; Bio-Rad, Hercules, Calif.) following the manufacturers instructions. Finally, the efficacy of the AONs was assessed by performing a PCR from exon 30 to exon 31 or a PCR spanning from exon 29 to 34.

B) Mutation: c.4539+2001G>A—Minigene

Generation of a Minigene

A minigene was created including part of intron 29, the complete exon 30, intron 30 and exon 31, and part of intron 31. This genomic region was cloned into a pCI-Neo-Rhodopsin vector using the Gateway System. The resulting vector (coined pCI-Neo-Rho-ABCA4-30-31 wild type, SEQ ID NO: 50) was used to introduce the c.4539+2001G>A mutation by site-directed mutagenesis (new vector was coined pCI-Neo-Rho-ABCA4-c.4539+2001A, SEQ ID NO: 53). Both control and mutated vectors were validated by Sanger sequencing. The minigenes were then transfected in HEK293T cells, which were harvested 48 h post-transfection and were subjected to RT-PCR analysis in order to detect the splicing defect.

AON Design and Testing

The transfection of minigene pCI-Neo-Rho-ABCA4-c.4539+2001A in HEK293T cells showed the insertion of the pseudoexon. Using the sequence of this pseudoexon, several AONs were designed. AONs were delivered together with the minigene in HEK293T cells. Transfected cells were subjected to RNA analysis.

RNA Analysis

Total RNA was isolated by using the NucleoSpin RNA Clean-up Kit (catalog no., 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturers protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the iScript cDNA synthesis kit (catalog no., 1708891; Bio-Rad, Hercules, Calif.) following the manufacturers instructions. Finally, the efficacy of the AONs was assessed by performing a PCR from exon 30 to exon 31 or a PCR spanning from exon 29 to 34.

C) Mutations: c.4539+2001G>A and c.4539+2028C>T—PPCs Assessment

Generation of Photoreceptor Precursor Cells (PPCs) Skin biopsies of a patient carrying the c.4539+2001G>A (M1) in a heterozygous manner and of a patient carrying the carrying the c.4539+2028C>T (M2) in a heterozygous state were obtained and fibroblast cell lines were generated. Subsequently, induced pluripotent stem cells (iPSCs) were reprogrammed as described previously (Sangermano et al., 2016), and differentiated to photoreceptor precursor cells (PPCs) using a method adapted from Sangermano et al. (2016) or from Flamier et al. (2016). Differentiated cells were subjected to RT-PCR analysis. ABCA4 Transcript Analysis

After thirty days of differentiation, control and patient-derived PPCs were harvested. Reverse transcription-PCR (RT-PCR) analysis was performed using primers located in exon 2 (forward) and exon 5 (reverse) or exon 30 (forward) and exon 31 (reverse) of the ABCA4 gene. Actin (ACTS) primers were used as a control. Primer sequences are represented by SEQ ID NOs: 54-77. All reaction mixtures (50 μl) contained 10 μM of each primer pair, Taq DNA Polymerase, 1 U/μl (cat. no. 11647679001, Roche, Basel, Switzerland), 10×PCR buffer without MgCl₂, 25 mM MgCl₂, 10 mM dNTPs, and 50 ng cDNA. PCR conditions were a first denaturation step of 94° C. for 5 min followed by 35 cycles of melting (94° C. for 30 s), annealing (58° C. for 30 s), and extension (72° C. for 1 min) steps, with a final elongation step of 72° C. for 5 min. PCR products were separated on a 1% (w/v) agarose gel and the resulting bands were excised and purified with the NucleoSpin® Gel & PCR cleanup kit (cat. no. 740609.250, Macherey-Nagel) according to manufacturer's protocol. Finally, 100 ng of the purified PCR product was analyzed via Sanger sequencing, in a 3100 or 3730 DNA Analyzer (Thermo Fisher Scientific).

Antisense Oligonucleotide (AON) Design

The sequence of the PE plus 50 base pairs flanking both sides were analyzed as described previously (Aartsma-Rus et al. 2012). Briefly, the overall RNA structure of the region of interest was analyzed with the mfold software (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form, last accessed 23 Jul. 2017), in order to identify partially open and closed regions. Splice enhancer motifs were determined using ESE finder 3.0 (http://krainer01.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home, last accessed 23 Jul. 2017). Special attention was paid to SC35 regions, as it has been demonstrated that there is a positive correlation between the presence of such motifs and the efficacy of AONs (Aartsma-Rus et al. 2012). Initially, this analysis led to the design of four AONs, two that overlap with the highest scoring SC35 motif (AON2 and AON3), one at the 5′-end of the PE (AON4) and one that overlaps with the c.4539+2001G>A mutation (AON1). At a later stage, 22 additional AONs were designed, to find correlations between the efficacy of AONs and their position towards the pseudoexon, their overlap with certain ESE motifs, and their specificity (i.e. whether single nucleotide mismatches could abolish their efficacy). The final AON sequences were also evaluated for the free energy of the molecule alone, the possibility to form dimers, and their interaction with the region of interest. For this, the RNA secondary structure tool (http://rna.urmc.rochester.edu/RNAstructureWeb, last accessed 23 Jul. 2017) was used, employing the RNA secondary structure and bifold prediction tools. We ensured that all AONs had a free energy value above −4 on their own, above −14 as a dimer and between 21 and 28 for the AON-region binding. This was calculated by using the estimated energy of the region of interest minus the energy of the AON bound to the region. All AON sequences had a length of 19 nucleotides with a Tm above 46° C. and a GC content between 40% and 65%. The sequences and properties of the AONs are listed in Table 1; further properties of the AONs for pseudoexon 30-31 (345) are listed in Table 2. AONs were chemically modified by adding a phosphorothioate backbone and a 2-O-methyl sugar modification 2OMe/PS to each nucleotide and were purchased from Eurogentec (Liège, Belgium). AONs were dissolved in PBS 1× (autoclaved twice) to a final concentration of 100 μM. Two sense oligonucleotides (SON-1 [SEQ ID NO: 280] and SON-2 [SEQ ID NO: 281]) were ordered with the same chemistry to be used as a negative control.

AON Treatment

Following differentiation, PPCs were treated with AONs (0.5 and 1 μM) by mixing the naked AONs directly with the culturing medium. After 24 h, cycloheximide (CHX, cat. no. C4859, Sigma Aldrich) was added at a final concentration of 0.1 mg/ml and cells were incubated for another 24 h. Forty-eight hours after AON delivery, cells were harvested, rinsed in PBS and RNA was isolated. cDNA synthesis was performed using 1 μg of RNA, as described above. All reactions were diluted to 20 ng/μl by adding 30 μl of distilled water. For RT-PCR analysis, 80 ng of cDNA was used for all the ABCA4 reactions whereas 40 ng for the ACTB analysis. All reaction mixtures (25 μl) contained 10 μM of each primer pair, Taq DNA Polymerase 1 UM (cat. no. 11647679001, Roche), 10×PCR buffer with MgCl2, supplemented with 1 mM MgCl2, 2 μM dNTPs, and 80 or 40 ng cDNA. PCR conditions for ABCA4 fragments from exon 30 to 31 were as follows: 94° C. for 2 min, 35 cycles of 30 s at 94° C., 30 s at 58° C. and 90 s at 72° C., followed by a final step of 2 min at 72° C. For actin amplification, PCR was performed under the same conditions except for an elongation time of 30 s. The entire volume of the ABCA4 PCR products and 10 μl of the actin amplicon were resolved on a 2% (w/v) agarose gel. The resulting bands were analyzed using Sanger sequencing. The ratio between correctly and aberrantly spliced variants was assessed by using Fiji software (Schindelin et al., 2012).

D) Mutations: c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T—Midigenes

Generation of a Midigene for Each Mutation

A midigene was created for each mutation (Sangermano et al. 2018). These midigenes include a considerable fragment of ABCA4 genomic DNA on each side of the corresponding mutations, often encompassing one or more of the flanking exons. This genomic region was cloned into a pCI-Neo-Rhodopsin vector using the Gateway System. The resulting vectors (pCI-Neo-Rho-ABCA4-intron6-intron7 wild type (SEQ ID NO: 290), pCI-Neo-Rho-ABCA4-intron6-intron11 wild type (SEQ ID NO: 292), pCI-Neo-Rho-ABCA4-intron11-intron15 wild type (SEQ ID NO: 295), pCI-Neo-Rho-ABCA4-intron29-intron32 wild type (SEQ ID NO: 297), pCI-Neo-Rho-ABCA4-intron31-intron37 wild type (SEQ ID NO: 300)) were used to introduce the c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T and c.5197-557G>T mutations to the corresponding vector by site-directed mutagenesis (new vectors were coined pCI-Neo-Rho-ABCA4-intron6-intron7 c.769-784T (SEQ ID NO: 291), pCI-Neo-Rho-ABCA4-intron6-intron11 c.859-540G (SEQ ID NO: 293), pCI-Neo-Rho-ABCA4-intron6-intron11 c.859-506C (SEQ ID NO: 294), pCI-Neo-Rho-ABCA4-intron11-intron15 c.1937+435G (SEQ ID NO: 296), pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539+1100G (SEQ ID NO: 298), pCI-Neo-Rho-ABCA4-intron29-intron32 c.4539+1106T (SEQ ID NO: 299) and pCI-Neo-Rho-ABCA4-intron31-intron37 c.5197-557T (SEQ ID NO: 301)). The control and mutated vectors were validated by Sanger sequencing. The midigenes were then transfected in HEK293T cells, which were harvested 48 h post-transfection and were subjected to RT-PCR analysis in order to detect the splicing defect.

AON Design and Testing

The RNA analysis of the HEK293T cells transfected with the midigenes, showed the pre-mRNA splicing defect that consisted of the insertion of a pseudoexon. Using the sequence of this pseudoexon several AONs were designed. Subsequently, AONs were transfected into HEK293T together with the midigenes. To validate the AON efficacy, cells were subjected to RT-PCR analysis. The efficiency of each of the AONs was assessed by delivering identical amounts of minigene and various concentrations of AON and performing RT-PCR analysis afterwards. For each experiment, one SON was included as a negative control. During the final check, we discovered that AON1 that was designed for the c.859-540C>G mutation was ordered incorrectly, and instead the sequence of AON3 for the c.5197-557G>T mutation was entered and provided. This also affects the interpretation of the results.

RT-PCR Analysis

Total RNA was isolated by using the NucleoSpin RNA Clean-up Kit (catalog no., 740955-50; Macherey-Nagel, Duren, Germany) according to the manufacturers protocol. RNA was quantified and cDNA was synthesized from 1 μg RNA by using the iScript cDNA synthesis kit (catalog no., 1708891; Bio-Rad, Hercules, Calif.) following the manufacturers instructions. Finally, the efficacy of the AONs was assessed by performing a PCR using the corresponding ABCA4 primers (SEQ ID NO: 302, Rhodopsin ex3 fw; SEQ ID NO: 303, ABCA4 ex7 rev; SEQ ID NO: 304, ABCA4 ex7 fw; SEQ ID NO: 305, ABCA4 ex8 rev; SEQ ID NO:306, ABCA4 ex13 fw; SEQ ID NO:307, ABCA4 ex14 rev; SEQ ID NO:308, ABCA4 ex30 fw; SEQ ID NO: 309, ABCA4 ex32 rev; SEQ ID NO: 310, ABCA4 ex32 tw; SEQ ID NO: 311, ABCA4 ex37 rev).

Results

A)

Minigene constructs harboring the c.4539+1100A>G or the c.4539+1106C>T mutation were transfected into HEK293T cells, together with a construct with the wild type ABCA4 sequence. As depicted in FIG. 1, both mutations result in the insertion of a 86-bp pseudoexon into the transcript (lanes marked with NT), although some remaining wild type transcript was also detected. Transfection of three different AONs showed that for both mutations, the pseudoexon insertion was completely abolished in the presence of AON1 (AON-1 Pseudoexon 30-31(68), SEQ ID NO: 15), and AON2 (AON-2 Pseudoexon 30-31(68), SEQ ID NO: 18), whereas AON3 (AON-3 Pseudoexon 30-31(68), SEQ ID NO: 21) resulted in a partial redirection of splicing events (FIG. 1). These data demonstrate the capability of AONs to redirect the aberrant splicing events due to the c.4539+1100A>G or the c.4539+1106C>T mutations.

B)

A minigene construct harboring the c.4539+2001A>G mutation (A) was transfected into HEK293T cells, together with a construct with the wild type ABCA4 sequence (G). Minigene construct harboring the c.4539+2001G>A mutation were transfected into HEK293T cells, together with a construct with the wild type ABCA4 sequence, RT-PCR analysis using RNA derived from these cells revealed the inclusion of a pseudoexon corresponding to a 345-bp sequence in intron 30, but only when cells were cultured in the presence of cycloheximide (+CHX), an agent regularly used to inhibit nonsense mediated degradation of aberrant transcripts. As shown in FIG. 3, all four AONs (AON1=AON-1 Pseudoexon 30-31(345), SEQ ID NO: 35, AON2=AON-2 Pseudoexon 30-31(345), SEQ ID NO: 38, AON3=AON-3 Pseudoexon 30-31(345), SEQ ID NO: 41, AON4=AON-4 Pseudoexon 30-31(345), SEQ ID NO: 44), redirected ABCA4 splicing completely, unlike the SON. Using the WT construct (left lane), as expected, only the normal, intact product without pseudoexon was detected. Herein AONs are interchangeably depicted as AON-n and AONn, wherein n is an integer; the AONs may be depicted with “-” or without “-”.

C)

In the photoreceptor precursor cells derived from a patient heterozygously carrying the ABCA4 c.4539+2001G>A (M1) mutation, RT-PCR analysis using RNA derived from these cells revealed the inclusion of a pseudoexon corresponding to a 345-bp sequence in intron 30, but only when cells were cultured in the presence of cycloheximide (+CHX), an agent regularly used to inhibit nonsense mediated degradation of aberrant transcripts. As illustrated in FIG. 2, upon transfection of four different AONs targeting this pseudoexon (AON1=AON-1 Pseudoexon 30-31(345), SEQ ID NO: 35, AON2=AON-2 Pseudoexon 30-31(345), SEQ ID NO: 38, AON3=AON-3 Pseudoexon 30-31(345), SEQ ID NO: 41, AON4=AON-4 Pseudoexon 30-31(345), SEQ ID NO: 44), the pseudoexon insertion completely disappeared after administration of AON1 and AON4. This was not the case for a negative control oligo SON (SEQ ID NO: 45), that has the complementary sequence of AON1, demonstrating that AON1 and AON4 effectively and specifically redirect aberrant splice events caused by the c.4539+2001G>A mutation.

To determine whether variants c.4539+2001G>A (M1) and c.4539+2028C>T (M2) result in aberrant splicing of ABCA4 pre-mRNA, fibroblast cell lines were generated from two unrelated Stargardt disease (STGD1) patients. A STGD1 patient with M1 carried the missense variant c.4892T>C (p.Leu1631Pro) in trans (Webster et al., 2001). A STGD1 patient with M2 carried the deep-intronic variant c.302+68C>T in cis, whereas a deletion c.6148-698_6670 delinsTGTGCACCTCCCTAG (p.?) was present on the other allele. In addition, a fibroblast line from a healthy control was generated. All cells were cultured in the absence and presence of cycloheximide (CHX), a compound used to suppress nonsense-mediated decay of RNA products carrying protein-truncating mutations. RT-PCR analysis with primers located in exons 30 and 31 revealed only one clear product, corresponding to the expected product encompassing exons 30 and 31 (FIG. 4). No aberrantly spliced products were detected in the fibroblasts from the STGD1 patients.

To investigate potentially retina-specific splicing defects caused by the two deep-intronic ABCA4 mutations, control and patient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) via lentiviral transduction of the Yamanaka factors (Takahashi et al., 2006). Quantitative PCR (q-PCR) (FIG. 7) and immunofluorescence analysis (data not shown) validated the pluripotency of the iPSCs. Subsequently, these iPSCs were differentiated for one month into photoreceptor precursor cells (PPCs). We used the protocol described previously by Flamier and colleagues (Flamier et al. 2016) to obtain a relatively homogeneous cone cell population, since the primarily affected cells in STGD1 are the cone photoreceptor cells. Characterization of control and patient-derived PPCs revealed a significantly increased expression of ABCA4, being ˜40 times higher in the control PPCs than control iPSCs, but only ˜3 times higher in M1- and M2-PPCs compared to M1- and M2-iPSCs. Further characterization of the PPCs revealed that all three cell lines were differentiated towards S-cones, although control PPCs expressed higher amounts of CRX and OPN1SW, compared to M1- and M2-PPCs (FIG. 7B).

As ABCA4 was robustly expressed in PPCs, we performed RT-PCR analysis from exon 30 to exon 31 which showed aberrant transcripts in both M1- and M2-derived PPCs upon CHX treatment, but not in control PPCs (FIG. 4A). Semi-quantification of the ratio between correctly and aberrantly spliced variants in the CHX-treated samples revealed that ˜25% of ABCA4 transcripts in the patient carrying M1 and ˜15% of ABCA4 transcripts in the patient carrying M2 were aberrant (FIG. 4B). A more detailed analysis of all bands by Sanger sequencing revealed a PE of 345 nt containing a premature stop codon (FIG. 5), which is predicted to result in the truncated protein product p.Arg1514Leufs*36. Interestingly, both variants included the same PE in the mRNA transcript upon CHX treatment. Once the sequence was identified, we studied the effect of both variants on splicing. According to all prediction software, neither M1 nor M2 changed the strength of the splice acceptor and donor site (FIG. 5). The splice donor site of the 345-nt PE contains ‘GC’ as canonical splice site sequence, which is only recognized by the Splice-Site-Finder-Like (SSFL) software. Further in silico predictions showed that M1 increases the strength of an exonic splicing enhancer SF2 site and creates a new SRp55 motif, whereas M2 creates one SC35 and two SRp40 motifs (FIG. 5).

Subsequent in-depth analysis of all the bands observed by RT-PCR revealed that one band contained heteroduplexes of the correctly spliced transcript together with the one containing the PE (FIG. 4). Moreover, an extra faint band lacking the last 73 bp of exon 30 was found in all samples treated with CHX, including the control. A relatively weak splice donor site (Human Splicing Finder (HSF) score: 75.9) explains this alternative transcript that was also detected in the heteroduplex band (FIG. 4). This splice product (r.4467_4539 del, p.Cys1490Glufs*12) was also identified as a result of non-canonical splice site variants at the ‘natural’ splice donor site of exon 30 (R. Sangermano, M. Khan et al. 2018). Interestingly, this new donor site was previously reported as a splice acceptor site (HSF score: 89.6) creating an isoform lacking the first 114 bp of exon 30 (Gerber et al., 1998).

In seven STGD1 cases with M2 in whom this was investigated, c.302+68C>T was found in cis (R. Allikmets, unpublished data; Braun et al., 2013; Lee et al., 2016 and Zernant et al., 2014). To study the contribution of this variant to STGD1 pathology, we performed RT-PCR of mRNA from control PPCs, M1- and M2-PPCs, treated and untreated with CHX, as well as from adult retina mRNA. As shown in FIG. 8, PCR primers located in exons 2 and 5 generated a canonical splice product of 459 nt, as well as a smaller fragment of 317 nt, in all PPCs and in human retina. Validation of the bands by Sanger sequencing revealed that the 317-nt fragment was lacking exon 3 (size: 142 bp). No other splice products were observed, indicating that the c.302+68C>T variant does not result in the activation of cryptic splice sites and/or exonic splice enhancers.

Once the molecular mechanism associated with M1 and M2 variants was elucidated, we aimed to design a therapeutic approach, based on splicing modulation, to skip the PE. An attractive and efficient method is the use of AONs, small RNA molecules that are able to enter the cell, bind to the pre-mRNA and modify the splicing pattern. In order to increase their binding affinity and avoid RNaseH activation (and therefore transcript degradation), we used 2-O-methyl-modified RNA AONs with phosphorothioate (2OMe/PS) backbones, as previously reported (Collin et al., 2012; Garanto et al., 2016; Gerard et al., 2012 and Slijkerman et al., 2016). In total we designed four AONs: two to block the SC35 motif with the highest score located at the 3′ end of the PE (AON2, AON3), one to block the second-highest-score SC35 at the 5′ of the PE (AON4), and one to block the newly created SRp55 motif due to M1 (AON1; FIG. 6A). In addition, a sense oligonucleotide (SON), complementary to AON1 and containing the same chemical modifications as the other AONs but not able to bind to the pre-mRNA, was designed in the same region. AONs and SON were delivered to ˜1-month differentiated-PPCs and after 48 h, the RNA was analyzed. As expected, CHX treatment increased the presence of aberrantly spliced transcript in the non-treated cells (FIGS. 6B and 6C). In addition, there were no differences between the non-treated and the SON-treated cells. We have demonstrated that the AONs are efficient in exon skipping. We found that AON4 was efficiently able to produce up to ˜75% PE skipping in both cell lines at two different concentrations (FIG. 6D), while AON1 was very efficient in the M1 cell line. AON2 showed variable efficacy, while AON3 was able to redirect splicing both at 0.5 μM and 1 μM (FIGS. 6B, 6C and 6D). One explanation for AON2 and AON3 showing such different behavior despite targeting the same region could be the AON properties (Table 1, Table 2). AON3 compared with AON2 has a low GC content and Tm, which might affect the stability and binding capacity, therefore explaining its low efficiency.

To further expand on our search for the most potent AON to redirect the splice defects caused by the c.4539+2001G>A mutation, we designed and tested 22 additional AONs (AON5-AON26, SEQ ID NO:'s: 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 238, 241, 244, 247, 250, 253 and 256, respectively), and assessed their capability to redirect ABCA4 splicing by preventing the inclusion of the 345-nt pseudoexon. Previously tested AONs1-4 (SEQ ID NO:'s 35/184, 38/187, 41/190 and 44/193, respectively) were taken along, as well as two sense oligonucleotides (SON1 [SEQ ID NO: 280] and SON2 [SEQ ID NO: 281]). The results are depicted in FIG. 9. Besides AONs 1 and 4, other effective AONs included AON9, AON10, AON14, AON17, AON18, AON22, AON23 and AON24. Moderately effective AONs include AON2, AON8, AON11, AON13, AON16, AON20 and AON21. Hardly or none effective AONs include AON3, AON5, AON6, AON7, AON12, AON15, AON19, AON25 and AON26. When comparing the properties of these AONs, a number of things become apparent:

-   -   i) AONs targeting regions outside the pseudoexon (AON5, AON6,         AON7, AON25 and AON26) are not capable of redirecting ABCA4         splicing.     -   ii) AONs that have a single mismatch to their target are not         effective, i.e. AON1 is mutation-specific for c.4539+2001G>A,         and does not redirect splicing in a patient with the         c.4539+2028C>T mutation (FIG. 6D). Likewise, AON15 is         mutation-specific for the pseudoexon with the c.4539+2028C>T         change and is not effective in correcting splicing defects due         to the c.4539+2001G>A mutation (FIG. 9).     -   iii) AONs that are effective for redirecting splicing often         harbor an SC35 motif (both effective and moderately effective         had 1.8 and 1.45 times more SC35 motifs on average when compared         to the poorly and non-effective AONs). No big differences were         observed for SF2 and SRp40 motifs. For the moderately effective         AONs, we detected 4 and 2.6 times enrichment of SRp55 motifs         when compared to the effective and the group of poorly effective         and non-effective AONs, respectively).     -   iv) On average there were no differences in the length of the         AONs that redirect splicing and those that did not. However, we         did observe that the melting temperature (Tm) was on average 2         and 3 degrees higher in the effective AONs when compared to the         moderately effective group and the group comprised of the poorly         effective and non-effective AONs.     -   v) Also on average, both effective and moderately effective AONs         showed a percentage of GC content higher than 54%, while the         average of the poorly effective or non-effective AONs was below         48%.     -   vi) We did not observe clear differences between those AONs         binding to predicted mixed regions with partially open and         partially closed regions to those binding to predicted either         closed or open regions.         D)

Midigene constructs harboring the c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T or c.5197-557G>T mutation were transfected into HEK293T cells, together with a construct with the corresponding wild type ABCA4 sequences. As depicted in FIG. 10, all mutations result in the insertion of a pseudoexon with variable length and to a variable degree (lanes marked with NT). Transfection of three different AONs, as well as one general SON for each mutation, showed that for all mutations, at least one AON was effective in rescuing the pseudoexon insertion associated with this mutation. Specifically, for c.769-784C>T, addition of AON1 and AON2 result in a decrease of the pseudoexon while AON3 partially rescues the splice defect. For c.859-540C>G, AON1 is not effective, AON2 is very effective, while AON3 partially rescues the splice defect. However, during the final check, we discovered that AON1 that was designed for the c.859-540C>G mutation was ordered incorrectly, and instead the sequence of AON3 for the c.5197-557G>T mutation was entered and provided. This also affects the interpretation of the results. Therefore, the negative result found for AON1 is expected because the actual AON that was used is not specific for the corresponding pseudexon, and therefore should not work. For c.859-506G>C, AON1 and AON3 result in a decrease of the transcript containing the pseudoexon, while AON2 is not effective. For c.1937+435C>G, all three AONs result in a decrease of the ABCA4 transcripts harboring the pseudoexon. For the c.4539+1100A>G and the c.4539+1106C>T mutations, AON1 and AON2 appear to be effective while AON3 clearly is not. Finally, or c.5197-557G>T mutation, all three AONs result in a decrease of the transcripts with the pseudoexon. Together, these data demonstrate the capability of AONs to redirect the aberrant splicing events due to the all deep-intronic ABCA4 mutations tested, with at least one AON for each pseudoexon being effective.

Discussion

In this study, we showed that two neighboring deep-intronic variants in ABCA4, c.4539+2001G>A and c.4539+2028C>T, result in retina-specific inclusion of a 345-nt pseudoexon (PE) in a proportion of ABCA4 transcripts. This PE, which is predicted to lead to protein truncation (p.Arg1514Leufs*36), was found as a low-abundance alternative splice form of ABCA4 when performing deep RNA sequencing of human macula RNA (Braun et al, 2013). RT-PCR product quantification revealed more PE insertion due to M1 than to M2. On the basis of the ocular phenotypes of STGD1 patients carrying M1, and the nature of the variants observed in trans in these patients, M1 was proposed to act as a severe variant (Bauwens et al, 2015; Bax et al, 2015; Braun et al, 2013). In contrast, based on our own observations, and the limited clinical data available for some STGD1 patients carrying M2 (Lee et al, 2016), we hypothesize that M2 acts as a mild to moderately severe variant. We thus would expect that the amount of mutant mRNA in the patient carrying M1, who carries a missense variant in trans, should be equal to the amount of correct product. This is not the case, yet this comparison is difficult, as smaller products amplify more effectively and NMD suppression may be incomplete. The PE insertion due to M2 is less prominent than that for M1, which is in agreement with its less severe character. However, we cannot exclude the possibility that other cis-acting variants missed during locus sequencing (Zernant et al, 2014) act in concert with these intron 30 variants. In addition, cell-type specific mechanisms may play a role, since both patient-derived PPC lines were less well differentiated than the control PPC line, indicating the possibility of a delay in the differentiation. This may have a significant influence on the amount of PE insertion. A clear example of the importance of retinal differentiation for PE recognition was described for the deep-intronic c.2991+1655A>G variant in CEP290. Whereas in lymphoblastoid and fibroblast cells of patients harboring this mutation homozygously, the ratio between correctly and aberrantly spliced CEP290 is ˜1:1 (Collin et al, 2012; Garanto et al, 2016; den Hollander et al, 2006), in iPSC-derived photoreceptor cells the amount of aberrantly spliced CEP290 was found to be drastically increased (˜1:4 ratio; Parfitt et al; 2016). This study not only revealed insights into why this mutation, despite a ubiquitous expression of CEP290, resulted in a non-syndromic retinal phenotype, but also demonstrated the enormous power of using iPSC-derived retinal cells from patients to study splice defects in a relevant cellular system.

Previous inherited retinal disease (IRD)-associated intronic variants have created new splice acceptor or donor sites that allowed the insertion of a PE (Braun et al, 2013; Bonifert et al, 2016; Webb et al, 2012; van den Hurk et al, 2003; Vache et al, 2012; Rio Frio et al, 2009; Naruto et al, 2015; Mayer et al, 2016; Liguori et al, 2016; den Hollander et al, 2006; Carss et al, 2017). To our knowledge, we are the first to report on the insertion of a PE that is not due to this mechanism but likely because of the creation of new ESE motifs in IRDs. Intronic regions are riddled with pairs of predicted splice acceptor and donor sites that theoretically could flank a PE. Upon the identification of additional PEs that are not activated through the creation of splice sites, it will be possible to determine the sequence motifs that render cryptic PEs into real PEs.

The M1- and M2-associated PE insertions were successfully blocked by several AONs. A M1-specific AON was only effective in the M1-cell line, and even with a doubled AON concentration, AON1 was still unable to correct the splice defect in the M2 cell line. In addition, a M2-specific AON that has a single mismatch to the PE sequence was not effective in a patient with M1. These results highlight the specificity of the sequence and the fact that a single nucleotide mismatch is enough to change the efficacy of an AON. The newly created SRp55 motif may play a crucial role in the detection of the PE. Given the fact that both variants activate the same PE and AON4 is able to skip the PE in both cases, this remains to be further elucidated. One of the limitations of AONs is that they bind to specific sequences and therefore it is not possible to test the same AON in animal models if there is no conserved DNA/RNA region, unless a model is created in which part of the human sequence is inserted at the orthologous position in the animal genome. However, it is already known that the 2OMe/PS chemistry and 2MOE (2-O-Methoxyethyl)/PS are not toxic for the eye as shown in several animal models (Garanto et al, 2016; Gerard et al, 2015; Murray et al, 2015). Furthermore, the first AON commercialized was used to treat the eye condition CMV-retinitis (Fomivirsen approved for CMV retinitis: first antisense drug. AIDS treatment news, 7 (1998). Thus, AON technology seems to be a safe and promising approach to treat eye disorders. Owing to the lack of animal models, the use of iPSC-derived photoreceptors appears to be a suitable alternative, although it still needs to be elucidated whether the function of ABCA4 protein can be restored following treatment of these cells.

In conclusion, by using patient-derived iPSC differentiated into S-cones, we were able to identify the molecular defect due to two recurrent neighboring deep-intronic variants underlying STGD1. The splice defect consisted of the insertion of a 345-nt PE which appears to be tissue-specific and is most likely caused by the presence of newly generated exonic splicing enhancers, instead of by the creation of novel splice sites. Moreover, an AON-based therapeutic approach was designed and tested, showing that one AON was able to redirect the splice defect in both mutated cell lines. Furthermore, a variant-specific AON was very effective against M1 but not M2, indicating that one single nucleotide mismatch can change the AON efficiency drastically. For several other deep-intronic mutations in ABCA4 (i.e. c.769-784C>T, c.859-540C>G, c.859-506G>C, c.1937+435C>G, c.4539+1100A>G, c.4539+1106C>T or c.5197-557G>T) we have shown that all result in the insertion of a pseudoexon. AONs were designed to block the inclusion of these pseudoexon, and for each pseudoexon, at least one AON was capable of significantly decreasing the amount of aberrant ABCA4 transcripts. Overall, these results highlight the potential of AONs as a therapeutic tool for Stargardt disease.

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The invention claimed is:
 1. An antisense oligonucleotide for redirecting splicing that is: complementary or substantially complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 30, or a part thereof; and wherein the antisense oligonucleotide comprises a 2′-O alkyl phosphorothioate antisense oligonucleotide, such as 2′-O-methyl modified ribose (RNA), 2′-O-ethyl modified ribose, 2′-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
 2. An antisense oligonucleotide for redirecting splicing according to claim 1, wherein the part that is complementary or substantially complementary to a polynucleotide with a nucleotide sequence consisting of SEQ ID NO: 10, 30, or a part thereof, has a length of from about 8 to about 40 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
 3. An antisense oligonucleotide for redirecting splicing according to claim 1 that has a length of from about 8 to about 100 nucleotides, preferably from about 10 to about 40 nucleotides, more preferably from about 14 to about 30 nucleotides, more preferably from about 16 to about 24 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.
 4. An antisense oligonucleotide for redirecting splicing according to claim 1, wherein said antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 15, 18, 21, 165, 168, 171, 35, 38, 41, 44, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, 241, 244, 247, 250, 253, 256, and
 259. 5. An antisense oligonucleotide for redirecting splicing according to claim 1, comprising at least one ribonucleotide.
 6. An antisense oligonucleotide for redirecting splicing according to claim 1, comprising at least one ESE (exon splice enhancer) motif.
 7. A viral vector expressing an antisense oligonucleotide for redirecting splicing according to claims claim 1 when placed under conditions conducive to expression of the exon skipping antisense oligonucleotide.
 8. A pharmaceutical composition comprising an antisense oligonucleotide for redirecting splicing according to claim 1 and a pharmaceutically acceptable excipient.
 9. A pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is for intravitreal administration and is dosed in an amount ranged from 0.05 mg and 5 mg of total antisense oligonucleotides for redirecting splicing per eye.
 10. A pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is for intravitreal administration and is dosed in an amount ranged from 0.1 and 1 mg of total antisense oligonucleotides for redirecting splicing per eye, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg of total antisense oligonucleotides for redirecting splicing per eye.
 11. A method for the treatment of an ABCA4-related disease or condition requiring modulating splicing of ABCA4 of an individual in need thereof, said method comprising contacting a cell of said individual with an antisense oligonucleotide for redirecting splicing as defined in claim
 1. 12. The method according to claim 11, wherein the ABCA4-related disease or condition is Stargardt disease. 